After graduating from the University of Melbourne in 1925, Eccles studied at the University of Oxford under a Rhodes scholarship. He received a Ph.D. there in 1929 after having worked under the neurophysiologist Charles Scott Sherrington. He held a research post at Oxford before returning to Australia in 1937, teaching there and in New Zealand over the following decades.

Eccles conducted his prizewinning research while at the Australian National University, Canberra (1951–66). He demonstrated that one nerve cell communicates (action potential) with a neighbouring cell by releasing chemicals into the synapse (the narrow cleft, or gap, between the two cells). He showed that the excitement of a nerve cell by an impulse causes one kind of synapse to release into the neighbouring cell a substance (probably acetylcholine) that expands the pores in nerve membranes. The expanded pores then allow free passage of sodium ions into the neighbouring nerve cell and reverse the polarity of electric charge. This wave of electric charge, which constitutes the nerve impulse, is conducted from one cell to another. In the same way, Eccles found, an excited nerve cell induces another type of synapse to release into the neighbouring cell a substance that promotes outward passage of positively charged potassium ions across the membrane, reinforcing the existing polarity and inhibiting the transmission of an impulse. (See also action potential.)

Eccles's research, which was based largely on the findings of Hodgkin and Huxley, settled a long-standing controversy over whether nerve cells communicate with each other by chemical or by electric means. His work had a profound influence on the medical treatment of nervous diseases and research on kidney, heart, and brain function.

Among his scientific books are Reflex Activity of the Spinal Cord (1932), The Physiology of Nerve Cells (1957), The Inhibitory Pathways of the Central Nervous System (1969), and The Understanding of the Brain (1973). He also wrote a number of philosophical works, including Facing Reality: Philosophical Adventures by a Brain Scientist (1970) and The Human Mystery (1979).