N-glycosylation usually occurs at the Asn-Xaa-Ser/Thr sequon of glycoproteins in Pichia pastoris, exerting great effects on expression efficiency; however, Asn-Xaa-Thr is more efficiently glycosylated than Asn-Xaa-Ser. In this study, the role of the two sequons in the expression of recombinant elastase (rPAE) was investigated. At N43, N212, and N280 of rPAE, Asn-Xaa-Thr was substituted for the native Asn-Xaa-Ser sequon through site-directed mutagenesis, and the two sequon forms were introduced into rPAE at N36 and N264. As expected, substitution at N36, N43, N212, and N280 enhanced the degree of N-glycosylation. At N212 or N280, substitution increased rPAE production effectively by 43 and 25 %, respectively. In comparison, at N36, N43, and N264, the change inhibited rPAE expression to varying extents; specifically, substitution at N36 resulted in a 31 % decrease, while substitution at N43 or N264 resulted in a decrease of less than 9 %. It is suggested that the effect of the substitution of Asn-Xaa-Thr for Asn-Xaa-Ser on rPAE expression is roughly related to the role of the original Asn-Xaa-Ser sequon. As the conversion of Ser to Thr at N-glycosylation sites through site-directed mutagenesis is easily achieved, it is a feasible means of improving the expression of recombinant proteins in P. pastoris.