Abstract: The cDNAs encoding chicken cystatin and its N-glycosylation-modified mutant (Asn106−Ile108→Asn106−Thr108) were cloned into the pGAPZαC expression vector, using the GAP as promoter and Zeocin as resistant agent, and transformed into Pichia pastoris X-33 expression host. The effect of N-glycosylation on the stability of recombinant chicken cystatin was investigated. A large quantity of recombinant chicken cystatin and the Asn106-glycosylated cystatins were expressed and secreted into broth using α-factor preprosequence. The Ki of the recombinant chicken cystatin (0.08 nM) was similar to that of wild-type chicken cystatin (0.05 nM). They acted as a competitive inhibition reaction against papain. According to the Ki, the inhibition ability of Asn106-glycosylated mutant cystatin (Ki = 9.5 nM) was weaker than that of the wild-type one. However, N-glycosylation at Asn106 substantially enhanced the freezing stability of recombinant chicken cystatin overexpressed in P. pastoris.