Glycerol-3-phosphate dehydrogenase is a biomedically important enzyme that plays a crucial role in lipid biosynthesis. It is activated by a ligand-gated conformational change that is necessary for the enzyme to reach a catalytically competent conformation capable of efficient transition state stabilization. While the human form (hlGPDH) has been the subject of extensive structural and biochemical studies, corresponding computational studies to support and extend the experimental observations have been lacking. We perform here detailed empirical valence bond and Hamiltonian replica exchange molecular dynamics simulations of wild-type hlGPDH and its variants, as well as providing a novel crystal structure of the binary hlGPDH·NAD R269A variant where the enzyme is present in the open conformation. We estimated the activation free energies for the hydride transfer reaction in wild-type and substituted variants of hlGPDH and investigated the effect of mutations on the catalysis from a detailed structural study. Our structural data and simulations also illustrate the critical role of the R269 side chain in facilitating the closure of hlGPDH into a catalytically competent conformation, through modulating the flexibility of a key catalytic loop (292-LNGQKL-297), thus rationalizing a tremendous 41,000-fold decrease experimentally in the turnover number, kcat, upon truncating this residue. Taken together, our data highlight the importance of this ligand-gated conformational change in catalysis, a feature that can be exploited both for protein engineering and for the design of novel allosteric inhibitors targeting this biomedically important enzyme.