The Penningtons profited well from the English Renaissance of Queen Elizabeth. Sir Isaac's father, Robert Pennington, was a member of the Worshipful Company of Fishmongers. That was the fourth of the 107 Livery Companies that controlled the economy and society of the City of London. Sir Isaac was also a Liveryman and 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. At his father's death, in 1627, he was knighted by the Speaker of the House of Commons. 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.

From 1642 to 1645 Penington was lieutenant of the Tower of London. In that capacity he was present during the execution of William Laud, the Archbishop of Canterbury and the fervent supporter of King Charles 1st and the Divine Right of Kings to rule. As a Commisoner for Cromwell's High Court of Justice, Sir Isaac participated in the treason trail of King Charles but did not sign Charles' death warrant. But he probably did vote for conviction for King Charles 2nd had him arrested and tried for treason and imprisoned. He died in the Tower, from the effects of ill-usage, 17 Dec 1661, before his sentence was put into execution.