Palin wrote most of his comedic material with fellow Python member Terry Jones. Before Monty Python, they had worked on other shows including the Ken Dodd Show, The Frost Report, and Do Not Adjust Your Set. Palin appeared in some of the most famous Python sketches, including "Argument Clinic", "Dead Parrot sketch", "The Lumberjack Song", "The Spanish Inquisition", "Bicycle Repair Man" and "The Fish-Slapping Dance". He also regularly played a Gumby.

Palin continued to work with Jones away from Python, co-writing Ripping Yarns. He has also appeared in several films directed by fellow Python Terry Gilliam and made notable appearances in other films such as A Fish Called Wanda (1988), for which he won the BAFTA Award for Best Actor in a Supporting Role. He also began a new career as a travel writer and travel documentarian in programmes broadcast on the BBC. His journeys have taken him across the world, including the North and South Poles, the Sahara, the Himalayas, Eastern Europe, and Brazil; in 2018, he visited North Korea, documenting his visit to the isolated country in a series broadcast on Channel 5.

In a 2005 poll to find The Comedians' Comedian, he was voted the 30th favourite by fellow comedians and comedy insiders. From 2009 to 2012 he was President of the Royal Geographical Society. On 12 May 2013, Palin was made a BAFTA fellow, the highest honour that is conferred by the organisation. Having been awarded a CBE for services to television in the 2000 New Year Honours, Palin received a knighthood in the 2019 New Year Honours for services to travel, culture and geography.