The role of oligosaccharide modification in human acid β-glucosidase function was investigated. This lysosomal enzyme has five putative N-glycosylation sites, four of which are occupied. The unglycosylated human protein was stable when expressed in bacteria or in Spodoptera frugiperda cells in the presence of tunicamycin but lacked catalytic activity. Deglycosylation of purified acidβ-glucosidase from human placenta with N-Glycanase^™ under native conditions resulted in the removal of an accessible oligosaccharide chain from a single site with no effect on activity, whereas studies demonstrate that occupancy of at least one glycosylation site is required for the formation and maintenance of acid β-glucosidase in an active conformation.