Objective To investigate the effect of glycosylation at Asn302 of pro-urokinase (pro-UK) on the stability in culture supernatant. Methods Nonglycosylated pro-UK was constructed by site-directed mutagenesis of Asn302 to Ala302.The pro-UK mutant and native pro-UK were transfected into dhfr~ - -CHO cells, and serum-free culture supernatant was harvested and incubated at 4℃ and 37℃, respectively. The pro-UK activity in culture supernatant was measured by the optical density (OD) increase with time (12 hours) at 405 nm. Without thermolysin activation, the percentage of single chain pro-UK was measured. Results After 48 hours of incubation at 4℃, the activities of pro-UK mutant and native pro-UK decreased 3.7% and 2.9% respectively, and at 37℃ decreased 37.9% and 23.5%, respectively. The total activity of native pro-UK was significantly higher than that of nonglycosylated mutant at 37℃. The single-chain percentage of native pro-UK was higher than that of nonglycosylated mutant at both 4℃ and 37℃. Conclusion Higher temperature increases the proteolysis of pro-UK. The glycosylation site on Asn302 is beneficial to pro-UK stability in culture supernatant. Objective To investigate the effect of glycosylation at Asn302 of pro-urokinase (pro-UK) on the stability in culture supernatant. Methods Nonglycosylated pro-UK was constructed by site-directed mutagenesis of Asn302 to Ala302.The pro-UK mutant and native pro -UK were transfected into dhfr ~ - CHO cells, and serum-free culture supernatant was harvested and incubated at 4 ° C and 37 ° C, respectively. The pro-UK activity in culture supernatant was measured by the optical density (OD) increase with Results after 48 hours of incubation at 4 ° C, the activities of pro-UK mutant and native pro-UK decreased by 3.7% and The total activity of native pro-UK was significantly higher than that of nonglycosylated mutant at 37 ° C. The single-chain percentage of native pro-UK was higher than 37.9% and 23.5% respectively. that of nonglycosylated mutant at b Conclusion Higher temperature increases the proteolysis of pro-UK. The glycosylation site on Asn302 is beneficial to pro-UK stability in culture supernatant.