The Mayflower was used primarily as a cargo ship, involved in active trade of goods (often wine) between England and other European countries, (principally France, but also Norway, Germany, and Spain). At least between 1609 and 1622, it was mastered by Christopher Jones, who would command the ship on the famous transatlantic voyage, and based in Rotherhithe, London, England. After the famous voyage of the Mayflower, the ship returned to England, likely dismantled for scrap lumber in Rotherhithe in 1623, only a year after Jones's death in March 1622. The Mayflower Barn, just outside the Quaker village of Jordans, in Buckinghamshire, England, is said to be built from these timbers.

Details of the ship's dimensions are unknown; but estimates based on its load weight and the typical size of 180-ton merchant ships of its day suggest an estimated length of 90–110 feet (27.4–33.5 m) and a width of about 25 feet (7.6 m). The ship was manned by a crew of 25-30.