Many studies have demonstrated that the properties of enzymes expressed in eukaryotes can be affected by the position and extent of glycosylation on enzyme. In this study, two potential glycosylation sites (the 8th and the 58th asparagine) were identified and the effect of propeptide glycosylation on Rhizomucor miehei lipase (RML) expressed in Pichia pastoris was investigated. To better understand the effect of glycosylation on the activity of RML, three mutants (M1, generated by N8A; M2, generated by N58A; and M3, generated by N8A and N58A) were designed to generate deglycosylated enzymes. The results showed that deglycosylated RML exhibited a twofold higher activity compared to the wild type. However, it was also found that glycosylation on the propeptide was important for the removal of the propeptide by Kex2 protease and secretion of the enzyme. Thus, our study provided a further understanding into the role of glycosylation on enzyme function.