The only son of King James IV of Scotland and his queen Margaret Tudor to survive infancy, he was born on 10 April 1512, at Linlithgow Palace, Linlithgowshire and was christened the next day, receiving the titles Duke of Rothesay and Prince and Grand Steward of Scotland. James had a claim to the English throne as his maternal grandfather was Henry VII of England.He was just seventeen months old when his father was killed at the Battle of Flodden Field on 9 September 1513. James was crowned in the Chapel Royal at Stirling Castle on 21 September 1513. During his childhood, the country was ruled by regents, first by his mother, the sister of King Henry VIII of England, until she remarried the following year, and thereafter by John Stewart, 2nd Duke of Albany, who was himself next in line to the throne after James and his younger brother, the posthumously-born Alexander Stewart, Duke of Ross. Other regents included Robert Maxwell, 5th Lord Maxwell, a member of the Council of Regency who was also bestowed as Regent of Arran, the largest island in the Firth of Clyde. In February 1517, James came from Stirling to Holyroodhouse, Edinburgh, but during an outbreak of plague in the city he was moved to the care of Antoine d'Arces at nearby rural Craigmillar Castle. At Stirling, the 10-year-old James had a guard of 20 footmen dressed in his colours, red and yellow. When he went to the park below the Castle, "by secret and in right fair and soft wedder (weather)," six horsemen would scour the countryside two miles roundabout for intruders

As early as August 1517, a clause of the Treaty of Rouen provided that if the Auld Alliance between France and Scotland was maintained, James should have a French royal bride. Yet the daughters of Francis I of France were promised elsewhere or sickly.[18] Perhaps to remind Francis of his obligations, James's envoys began negotiations for his marriage elsewhere from the summer of 1529, both to Catherine de'Medici, the Duchess of Urbino, and Mary of Austria, Queen of Hungary, the sister of Holy Roman Emperor Charles V. But plans changed. In February 1533, two French ambassadors, Guillaume du Bellay, sieur de Langes, and Etienne de Laigue, sieur de Beauvais, who had just been in Scotland, told the Venetian ambassador in London that James was thinking of marrying Christina of Denmark.[19] Francis I insisted that his daughter Madeleine's health was too poor for marriage. Eventually, on 6 March 1536, a contract was made for James V to marry Mary of Bourbon, daughter of the Duke of Vendôme. She would have a dowry as if she were a French Princess. James decided to visit France in person. He sailed from Kirkcaldy on 1 September 1536, with the Earl of Argyll, the Earl of Rothes, Lord Fleming, David Beaton, the Prior of Pittenweem, the Laird of Drumlanrig and 500 others, using the Mary Willoughby as his flagship.[20] First he visited Mary of Bourbon at St. Quentin in Picardy, but then went south to meet King Francis I.[21] During his stay in France, in October 1536, James went boar-hunting at Loches with Francis, his son the Dauphin, the King of Navarre and Ippolito II d'Este.[22] James renewed the Auld Alliance and fulfilled the 1517 Treaty of Rouen on 1 January 1537 by marrying Madeleine of Valois, the king's daughter, in Notre Dame de Paris. The wedding was a great event: Francis I made a contract with six painters for the splendid decorations, and there were days of jousting at the Louvre.[23] At his entry to Paris, James wore a coat described as "sad cramasy velvet slashed all over with gold cut out on plain cloth of gold fringed with gold and all cut out, knit with horns and lined with red taffeta."[24] James V so liked red clothing that, during the wedding festivities, he upset the city dignitaries who had sole right to wear that colour in processions. They noted he could not speak a word of French.[25] When James was at Compiègne on 25 February 1537, a messenger from Pope Paul III arrived with a gift of a sword and hat.[26] James and Madeleine returned from France on 19 May 1537, arriving at Leith, the king's Scottish fleet accompanied with ten great French ships.[27] As the couple sailed northwards, some Englishmen had come aboard off Bridlington and Scarborough. While the fleet was off Bamburgh on 15 May, three English fishing boats supplied fish, and the King's butcher landed in Northumbria to buy meat.[28] The English border authorities were dismayed by this activity.[29] Madeleine did not enjoy good health. In fact, she was consumptive and died soon after arrival in Scotland in July 1537. Spies told Thomas Clifford, the Captain of Berwick, that James omitted "all manner of pastime and pleasure," but continually oversaw the maintenance of his guns, going twice a week secretly to Dunbar Castle with six companions.[30] James then proceeded to marry Mary of Guise, daughter of Claude, Duke of Guise, and widow of Louis of Orleans, Duke of Longueville, by proxy on 12 June 1538. Mary already had two sons from her first marriage, and the union produced two sons. However, both died in April 1541, just eight days after baby Robert was baptised. Their daughter and James's only surviving legitimate child, Mary, was born in 1542 at Linlithgow Palace.

The death of James's mother in 1541 removed any incentive for peace with England, and war broke out. Initially the Scots won a victory at the Battle of Haddon Rig in August 1542. The Imperial ambassador in London, Eustace Chapuys, wrote on 2 October that the Scottish ambassadors ruled out a conciliatory meeting between James and Henry VIII in England until the pregnant Mary of Guise delivered her child. Henry would not accept this condition and mobilised his army against Scotland.[51] James was with his army at Lauder on 31 October 1542. Although he hoped to invade England, his nobles were reluctant.[52] He returned to Edinburgh on the way writing a letter in French to his wife from Falahill mentioning he had three days of illness.[53] Next month his army suffered a serious defeat at the Battle of Solway Moss. He took ill shortly after this, on 6 December, by some accounts this was a nervous collapse caused by the defeat, although some historians consider that it may just have been an ordinary fever. John Knox later described his final movements in Fife.[54] Whatever the cause of his illness, he was on his deathbed at Falkland Palace when his only surviving legitimate child, a girl, was born. Sir George Douglas of Pittendreich brought the news of the king's death to Berwick. He said James died at midnight on Thursday 15 December; the king was talking but delirious and spoke no "wise words." According to George Douglas in his delirium James lamented the capture of his banner and Oliver Sinclair at Solway Moss more than his other losses.[55] An English chronicler suggested another cause of the king's grief was his discomfort on hearing of the murder of the English Somerset Herald, Thomas Trahern, at Dunbar.[56] James was buried at Holyrood Abbey in Edinburgh. Before he died, he is reported to have said, "it came wi a lass, it'll gang wi a lass" (meaning "It began with a girl and it will end with a girl"). This was either a reference to the Stewart dynasty's accession to the throne through Marjorie Bruce, daughter of Robert the Bruce or to the medieval origin myth of the Scots nation, recorded in the Scotichronicon in which the Scots people are descended from the Princess Scota.