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Kettlewell was educated at Cambridge University and St. Bartholomew's Hospital, London, where he gained his medical qualification in 1933. He practiced in Cranleigh and then worked as an anesthetist in Surrey. After the war he worked in South Africa at the International Locust Control Centre in Cape Town before returning to Britain in 1952 as research fellow in genetics at Oxford, a post he continued to hold until his retirement in 1974.

Kettlewell is best known for his work on the occurrence of melanism – black pigmentation in the epidermis of animals. In 1953 he set out to explain why, in the mid-19th century, certain moth species had a light coloration, which camouflaged them on such backgrounds as light tree trunks where they sat motionless during the day. However by the 1950s, of 760 species of larger moths in Britain 70 had changed their light color and markings for dark or even totally black coloration.

Kettlewell suspected that the success of the melanic form was linked with the industrial revolution and the consequent darkening of the trees by the vast amounts of smoke produced by the 19th-century factories. To test his hypothesis he released large numbers of the dark and light forms of the peppered moth, Biston betularia, in the polluted woods around Birmingham and in a distant unpolluted forest. As many of the released moths as possible were recaptured and when the results were analyzed it was found that the light form had a clear advantage over the dark in the unpolluted forest but in the polluted Birmingham woods the result was just the opposite. From this Kettlewell concluded that if the environment of a moth changes so that it is conspicuous by day, then the species is ruthlessly hunted by predators until it mutates to a form better suited to its new environment. His work was seen as a convincing and dramatic confirmation of the Darwinian hypothesis of evolution through natural selection.