Born in Melbourne, Australia, Eccles was educated at the university there and at Oxford University. In Oxford he worked with Charles Sherrington on muscular reflexes and nervous transmission across the synapses (nerve junctions) from 1927 to 1937. He then worked in Australia at the Institute of Pathology from 1937 to 1943. After a period in New Zealand, as professor of physiology at the University of Otago from 1944 to 1951, Eccles returned to Australia to the Australian National University, Canberra, where he served as professor of physiology from 1951 to 1966. In 1966 Eccles moved to the USA, working first in Chicago and finally, from 1968 until his retirement in 1975, at the State University of New York, Buffalo.

While at Canberra Eccles carried out work on the chemical changes that take place at synapses, pursuing the findings of Alan Hodgkin and Andrew Huxley, with whom he subsequently shared the 1963 Nobel Prize for physiology or medicine. Eccles showed that excitation of different nerve cells causes the synapses to release a substance (probably acetylcholine) that promotes the passage of sodium and potassium ions and effects an alternation in the polarity of the electric charge. It is in this way that nervous impulses are communicated or inhibited by nerve cells. Eccles was the author of Reflex Activity of the Spinal Cord (1932) and The Physiology of Nerve Cells (1957).

After his retirement Eccles published a number of works on the mind-body problem. Notable among them are The Self and the Brain (1977), written in collaboration with Karl Popper, The Human Mystery (1979), and The Creation of the Self (1989).