John Mifflin was born in Philadelphia, the son of George Mifflin and Esther Cordery. He and his first wife, Elizabeth Bagnell, were the parents of seven children, including Thomas Mifflin, A.B. 1760, governor of Pennsylvania (1788-1799) and Penn trustee (1773-1791). With his second wife, Sarah Fishbourne, Mifflin had three children, including John Fishbourne Mifflin, A.B. 1775 and Penn trustee (1802-1813).
A wealthy Quaker merchant, John Mifflin held numerous significant political posts including that of provincial councilor as well as of councilman, alderman, and justice of the peace.
John Mifflin was a trustee of the College and Academy of Philadelphia (now the University of Pennsylvania) from 1755 until his death in 1759.