Abstract: The aim of this study was to investigate the effect of cholesterol on the level of tyrosine phosphorylation of Inner Mongolian cashmere goat semen during cryopreservation. In the experiment, the spermatozoa were treated with different concentrations（1 mg/mL, 2 mg/mL, 3 mg/mL and 4 mg/mL） of cholesterol-cyclodextrin（CLC） before cryopreservation, the viability of frozen-thawed spermatozoa was detected using the computer-assisted semen analysis system（CASAS）, the DNA integrity, acrosome and plasma membrane integrity, and free Ca²⁺ level and the sperm protein tyrosine phosphorylation level of frozen-thawed spermatozoa were detected by flow cytometry and Western blot, respectively. The results showed that the CLC supplementation significantly improved the viability, acrosome and plasma membrane integrity（P＜0.05） and the DNA integrity of frozen-thawed sperms in CLC groups（P＜0.01）, compared with the control group; The free Ca²⁺ levels in the sperms of the 2 mg/mL CLC group were significantly lower than that of the control group（P＜0.05）; the CLC treatment significantly reduced the protein tyrosine phosphorylation level of 30 kD、 37 kD 、40 kD in frozen-thawed sperms. It could be deduced that the CLC supplementation in Inner Mongolian cashmere goat semen before cryopreservation could reduce the level of sperm protein tyrosine phosphorylation, and improve the cryosurvival of sperms.