© CSIRO 2014. Neutron crystallography allows direct localization of hydrogen positions in biological macromolecules. Within enzymes, hydrogen atoms play a pivotal role in catalysis. Recent advances in instrumentation and sample preparation have helped to overcome the difficulties of performing neutron diffraction experiments on protein crystals. The application of neutron macromolecular crystallography to a growing number of proteins has yielded novel structural insights. The ability to accurately position water molecules, hydronium ions, and hydrogen atoms within protein structures has helped in the study of low-barrier hydrogen bonds and hydrogen-bonding networks. The determination of protonation states of protein side chains, substrates, and inhibitors in the context of the macromolecule has provided important insights into enzyme chemistry and ligand binding affinities, which can assist in the design of potent therapeutic agents. In this review, we give an overview of the method and highlight advances in knowledge attained through the application of neutron protein crystallography.