Glycosylation is one of the post-translational modifications, and known to be important for protein folding, stability, and secretion. When nascent glycoproteins enter the ER, a preformed oligosaccharide known as dolichol-phosphate precursor is often attached co-translationally to certain Asn residue(s) involved in the consensus sequence Asn-Xaa-Ser/Thr (where Xaa represents any amino acid except Pro). Changes in glycosylation are early indicators of cellular changes in many diseases, most notably cancer, providing useful diagnostic markers and insights into disease progression and pathogenesis. Cathepsin L2, also known as cathepsin V, is a lysosomal cysteine protease that belongs to cathepsin family, and related to cancer invasion and metastasis. All cathepsin family members have potential N-glycosylation site(s), but role of N-glycosylation on cathepsin functions has not been fully understood. Cathepsin L2 contains two predicted N-glycosylation sites, Asn221 and Asn292, but glycosylation of cathepsin L2 has not been reported. We demonstrated that cathepsin L2 was N-glycosylated at both Asn221 and Asn292 sites using mass spectrometry and site-directed mutagenesis analyses. N-glycosylation of cathepsin L2 was found to be important for transportation to lysosome and secretion. Furthermore, N-glycosylation of cathepsin L2 was required for the enzymatic activity of cathepsin L2. These data demonstrated that functions of cathepsin L2 were controlled by N-glycosylation. Thus, inhibition of cathepsin L2 N-glycosylation may suppress cancer invasion and metastasis.