Enterokinase is an ideal tool protease for cleaving fusion proteins in genetic engineering. The bovine enterokinase light chain (bEK(L)) produced in Pichia pastoris was shown to be a glycoprotein. To study the effects of N-glycosylation on the biochemical properties of bEK(L), the enzyme was deglycosylated via site-directed mutagenesis. The results showed that elimination of the N-glycosylation sites of bEK(L) (N64, N103 and N165) did not significantly affect the protein secretion level in P. pastoris, but it does greatly influence its enzymatic activity. The N64Q increased the specific activity of the enzyme for GD(4)K-beta-naphthylamide and improved its catalytic efficiency. Moreover, the glycosylated bEK(L), is more thermostable than its deglycosylated counterparts. Structural analysis of glycosylated and deglycosylated bEK(L) revealed that the removal of N-glycosylation did not have pronounced changes on the secondary structure but there was a significant difference in the tertiary structure. In conclusion, this study demonstrated that the effects of glycosylation at different degrees and sites in bEK(L) were diverse. Moreover, this work will provide theoretical support for designing enzymes on the basis of N-glycosylation to meet the demands of the biochemical industry.