[2] In 1608 Guy and other members of the Society of Merchant Venturers, decided to act upon the letter received by the mayor from Chief Justice Sir John Popham concerning the colonisation of Newfoundland.

Guy visited the island in 1608 to scout possible locations for a settlement, selecting Cuper's Cove (present day Cupids, Newfoundland and Labrador) as the site of the colony.

On 27 April 1610 James I granted a charter to Henry Howard, 1st Earl of Northampton, Keeper of the Privy Seal, and others including John Guy and his brother Philip Guy, which incorporated them as the "Treasurer and Company of Adventurers and Planters of the Cities of London and Bristol, for the purpose of colonising Newfoundland, and comprehending as their sphere of action the southern and eastern parts of the new found land between 46° and 52° N. L."[2] Guy was appointed governor in 1610 by the London and Bristol Company and arrived at Cuper's Cove in August of that year with colonists, grain and livestock, after a quick passage of 21 days.

In 1612, the actions of the English pirate Peter Easton convinced Guy to abandon a second colony established at Renews in the spring of that year and strengthen the fortifications at Cuper's Cove.

The colonists built a boat called the Endeavour, which Guy used to lead a voyage into Trinity Bay in the Autumn of 1612 in an attempt to contact and establish a fur trade with the Beothuk, the native inhabitants of the island.

On 6 November, Guy's party met, shared a meal and exchanged gifts with a group of Beothuk somewhere in Bull Arm, Trinity Bay.

Five years later a visitor to Newfoundland wrote that the Bristol citizens had "planted a large circuit of the country, and builded there many fine houses, and done many other good services" Guy became disillusioned due to the lack of support from the London merchants, and remained in Bristol, though he later received a grant of land in Newfoundland which he named Sea Forest, and which he divided amongst his younger sons in his will in 1624.

John Guy in his capacity as an admiral in the Royal Navy fought against Turkish pirates operating in the English Channel during 1620, for which the grateful merchants of Bristol, provided him with a sum of money as a vote of thanks for his efforts.

Guy was actively involved in the House of Commons - sitting on Committees and introducing a private Members Bill to reduce interest rates by 2% from 10% to 8%.

The will of his son John (who died in 1640, from the plague which he caught whilst studying at the Middle Temple) calls for the erection of a monument for his father in St Stephen's Church.