Lysosomal β-hexosaminidase A (Hex A) is essential for the degradation of G_M2 gangliosides in the central and peripheral nervous system. Accumulation of G_M2 leads to severely debilitating neurodegeneration associated with Tay-Sachs disease (TSD), Sandoff disease (SD) and AB variant. Here, we present the X-ray crystallographic structure of Hex A to 2.8Å resolution and the structure of Hex A in complex with NAG-thiazoline, (NGT) to 3.25Å resolution. NGT, a mechanism-based inhibitor, has been shown to act as a chemical chaperone that, to some extent, prevents misfolding of a Hex A mutant associated with adult onset Tay Sachs disease and, as a result, increases the residual activity of Hex A to a level above the critical threshold for disease. The crystal structure of Hex A reveals an αβ heterodimer, with each subunit having a functional active site. Only the α-subunit active site can hydrolyze G_M2 gangliosides due to a flexible loop structure that is removed post-translationally from β, and to the presence of αAsn423 and αArg424. The loop structure is involved in binding the G_M2 activator protein, while αArg424 is critical for binding the carboxylate group of the N-acetyl-neuraminic acid residue of G_M2. The β-subunit lacks these key residues and has βAsp452 and βLeu453 in their place; the β-subunit therefore cleaves only neutral substrates efficiently. Mutations in the α-subunit, associated with TSD, and those in the β-subunit, associated with SD are discussed. The effect of NGT binding in the active site of a mutant Hex A and its effect on protein function is discussed.