In April 1640 Penington was elected a Member of Parliament (MP) for the City of London in the Short Parliament. He was re-elected MP for City of London for the Long Parliament in November 1640 and sat until 1653. On 16 August 1642 Parliament appointed him Lord Mayor of London after removing the Royalist Sir Richard Gurney, 1st Baronet from the position. He became a Colonel of the White Regiment in 1642 and from 1642 to 1645 he was Lieutenant of the Tower of London. In that capacity he was present during the execution of William Laud. He became Governor of the Levant Company in 1644, retaining the position to 1654.