Mannostatin A is a potent inhibitor of the mannose trimming enzyme Golgi α-mannosidase II (GMII), which acts late in the N-glycan processing pathway. Inhibition of this enzyme provides a route to blocking the transformation–associated changes in cancer cell surface oligosaccharide structures. In this paper, we report on the synthesis of new Mannostatin derivatives and analyze their binding in the active site of Drosophila GMII by X-ray crystallography. The results indicate that the interaction with the backbone carbonyl of Arg876 is crucial to the high potency of the inhibitor, an effect enhanced by the hydrophobic interaction between the thiomethyl group and an aromatic pocket vicinal to the cleavage site. The various structures indicate that differences in the hydration of protein-ligand complexes are also important determinant of plasticity as well as selectivity of inhibitor binding.