Penington was the oldest son of Isaac Penington, a Puritan who had served as the Lord Mayor of London. Penington married a widow named Mary Springett and they had five children. Penington's stepdaughter Gulielma Springett married William Penn. Convinced that the Quaker faith was true, Penington and his wife joined the Friends in 1657 or 1658. Penington became an influential promoter and defender of the Quaker movement, writing extensively on many topics. His writings are prized for their insightful and eloquent exploration of spiritual experience, with his Letters being read continuously within Quakerism for their spiritual counsel. His complete works were first published in 1681 and are still in print today.
Penington became an influential promoter and defender of the Quaker movement, publishing several books about it. He was imprisoned six times for his beliefs, starting in 1661. Sometimes the charge was refusal to take an oath, which went against Quaker teachings (see Testimony of Integrity), but was prohibited by the Quaker Act of 1662. At other times Penington was charged with attending a Quaker meeting, which was forbidden by the Conventicle Act of 1664.
Penington's wife, Mary, was a remarkable woman in her own right. Her daughter Gulielma, from her first marriage, to Sir William Springett (who died young), became the first wife of WIlliam Penn.