Kettlewell was born in Howden, Yorkshire, and educated at Charterhouse School. During 1926 he studied medicine and zoology at Gonville and Caius College, Cambridge. During 1929 he began clinical training at St Bartholomew's Hospital, London, then during 1935 joined a general medical practice in Cranleigh, Surrey. He also worked as an anesthetist at St. Luke's Hospital, Guildford. During World War II, from 1939 to 1945, he worked for the Emergency Medical Service at Woking War Hospital.
He emigrated to South Africa during 1949, and from then until 1954 was a researcher at the International Locust Control Centre at Cape Town University, investigating methods of locust control and going on expeditions to the Kalahari Desert, the Knysna Forest, the Belgian Congo, and Mozambique.
During 1952 he was appointed to a Nuffield Research Fellowship in the Department of Genetics of the Department of Zoology at Oxford University. Until 1954 he divided his time between South Africa and Oxford, then he gained the position of Senior Research Officer of the Department of Genetics and spent the rest of his career in Oxford as a genetics researcher. He was assigned to investigate peppered moth evolution under the supervision of E. B. Ford.
His grant was to study industrial melanism in general and in particular the peppered moth Biston betularia which had been studied by William Bateson during the 1890s. Kettlewell's research from three surveys between 1952 and 1972 seemed to show a static pattern with a high frequency of the dark-coloured carbonaria phenotype in industrial regions, and the light coloured typica moths the most common in more rural areas. In the first of Kettlewell's experiments moths were released into an aviary to observe how insectivorous birds reacted. He showed that the birds ate the moths, and found that if the camouflage of the moths made them difficult for him to see against a matching background, the birds too had difficulty in finding the moths. Most famously he then performed experiments involving releasing and then recapturing marked moths in polluted woodlands in Birmingham, and in unpolluted rural woods at Deanend Wood, Dorset, England. He demonstrated experimentally the efficiency of natural selection as an evolutionary force: light-coloured moths are more conspicuous than dark-coloured ones in industrial areas, where the vegetation is darkened by pollution, and are therefore easier prey for birds, but are less conspicuous in unpolluted rural areas, where the vegetation is lighter in colour, and therefore survive predation better. His experiment resulted in better understanding of industrial melanism and its effects on the evolution of species.
J.B.S. Haldane was of the opinion that Kettlewell had attempted to capitalize on Haldane's own observations, made as early as 1924, of the statistical probability of rate of change from light to melanic forms of the peppered moth. In 1961, Haldane and his graduate student (and later wife) Helen Spurway told Canadian lepidopterist Gary Botting that they questioned Kettlewell's data since it too "nicely" approximated Haldane's 1924 statistical calculations. Botting and Haldane at that time shared the opinion that some genetic mechanism other than bird predation was at work.