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Glycosylation plays a crucial role in glycoprotein stability and its correct folding. Murine acid sphingomyelinase (ASM) is a lysosomal glycoprotein. We studied the functional role of its individual N-linked oligosaccharides needed to maintain enzymatic activity and protein stability. Mutagenized cDNA constructs were heterologously expressed. All six potential N-glycosylation sites were modified. Incomplete glycosylation of the most distant C-terminal site resulted in two isoforms. Oligosaccharides at N-84, N-173, and N-611 were found to be of minor importance for enzymatic activity. The glycosylation defect at N-333 or N-393 reduced the enzymatic activity to 40% and at N-518 to less than 20%. These mutations did not effect the Km value. Glycosylation at N-333 and N-393 mainly contributed to the enzyme stability and prevented degradation at lysosomal acidic pH, whereas the low residual enzymatic activity of mutant ASM deficient in glycosylation at N-518 was caused by protein misfolding. The mutant protein was also prone to proteolysis when trapped in the endoplasmic reticulum/cis-Golgi after brefeldin A application. Insufficiently glycosylated ASM formed a stable complex with BiP, an immunoglobulin heavy chain-binding protein, and thus remained in the endoplasmic reticulum. 32PO4 labeling revealed that the glycosylation mutants of ASM were phosphorylated predominantly at mannose residues of oligosaccharides linked to N-84, N-333, and N-393.