Penington was the son of a fishmonger named Robert Penington, following him in becoming a Liveryman of the Worshipful Company of Fishmongers. He inherited several estates from his father and purchased one of his own. He made a fortune as a wine and cloth merchant. From 1626 he acted as finanacial agent to his second cousin, Admiral John Penington. He increased his commercial holdings in 1629 by becoming a partner in the brewery business of his second wife's family. His wife, Mary (nee Wilkinson), and he were both staunch Puritans.
In 1638 Penington became an alderman and the high sheriff of London. In 1640 he was elected a MP in the Short and Long Parliaments, representing the city of London. On August 16, 1642 Parliament appointed him Lord Mayor of London after removing the Royalist Sir Richard Gurney from the position.
In January 1649, he was appointed a commissioner of the High Court of Justice at the trial of King Charles, but he was not one of the signatories of the King's death warrant. He served on the Rump's council of state and on several government committees. He was made a knight in 1649.