Cronin, who was born in Chicago, Illinois, was educated at the Southern Methodist University and at Chicago, where he obtained his PhD in 1955. After a period at the Brookhaven National Laboratory he moved to Princeton in 1958 and later served as professor of physics from 1965 until 1971 when he was appointed to a comparable chair at the University of Chicago.

In 1956 Tsung Dao Lee and Chen Ning Yang made the startling claim that parity (P) was not conserved in weak interactions. To the surprise of physicists their bold conjecture was confirmed in a matter of months. It was however widely assumed that in a reaction the combination of parity and a property called charge conjugation (c) was conserved. Cronin and Val Fitch, together with James Christenson  and René Turlay, tested this CP conservation in 1964 by investigating the decay of neutral kaons. It was known that one type of kaon could decay into two pions; the other could not without violating CP conservation. Cronin and his colleagues discovered a small number of decays of the second type into two pions, clearly demonstrating that CP is violated.

The result is of fundamental interest for it is known that the combined properties of charge conjugation (c), parity (P), and time (T) are conserved – so that if CP is violated then the decay of the kaons is not symmetrical with respect to time reversal.

Cronin and Fitch shared the 1980 Nobel Prize for physics for this work.