Walker was educated at Oxford University, gaining his DPhil in 1969. In 1974 he joined the staff of the Medical Research Council at the Molecular Biology Laboratory, Cambridge.

In the 1970s Paul Boyer had proposed a theoretical model by which the enzyme ATP-synthase operating in mitochondria could catalyze the production of the adenosine triphosphate molecule (ATP), the main source of cellular energy. The model was partially verified by Walker in 1994. He determined the structure of the enzyme, first by low-resolution electron microscopy and eventually, using x-ray crystallography, constructed a three-dimensional model of the enzyme.

For his contribution to this field Walker shared the 1997 Nobel Prize for chemistry with Paul Boyer and Jens Skou.