In the Caribbean he sold them to the Spanish for 'hides, ginger, sugars, and some quantity of pearls, but he freighted also two other hulks with hides and like commodities.'

In 1567, on his last slaving voyage, in which his younger kinsman Francis Drake accompanied him, all but two of his ships and some treasure were seized by the Spanish in the port of San Juan de Uloa, Mexico, though he escaped and was later able to recover some of his captured men from Spain. In 1572 he became MP for Plymouth and in 1577 Treasurer of the Navy, in which role he greatly improved construction of ships. He was a senior commander in the Armada campaign, during which he was knighted, and subsequently joint commander with Frobisher in an expedition against the Portuguese coast in 1590. After the Armada he set up the 'chest at Chatham', the Naval welfare fund for seamen and in 1592 the hospital (alms-house) which still bears his name there. Hawkins died in Puerto Rico, shortly before Drake, on their final expedition to the West Indies in 1595–1596.

This portrait is inscribed 'Anno D[omi]ni 1581' , 'Aetatis Suae 44' and 'Sr John Hawkins', though if the age stated is correct it would have been painted in 1576.