Inducible nitric oxide synthase (iNOS) plays critical roles in the inflammatory response and host defense. Previous research on iNOS regulation mainly focused on its gene expression level, and much less is known about the regulation of iNOS function by N-glycosylation. In this study, we report for the first time that iNOS is N-glycosylated in vitro and in vivo. Mass spectrometry studies identified Asn695 as an N-glycosylation site of murine iNOS. Mutating Asn695 to Gln695 yields an iNOS that exhibits greater enzyme activity. The essence of nitric oxide synthase catalytic reaction is electron transfer process, which involves a series of conformational changes, and the linker between the flavin mononucleotide-binding domain and the flavin adenine dinucleotide-binding domain plays vital roles in the conformational changes. Asn695 is part of the linker, so we speculated that attachment of N-glycan to the Asn695 residue might inhibit activity by disturbing electron transfer. Indeed, our NADPH consumption results demonstrated that N-glycosylated iNOS consumes NADPH more slowly. Taken together, our results indicate that iNOS is N-glycosylated at its Asn695 residue and N-glycosylation of Asn695 might suppress iNOS activity by disturbing electron transfer.