Aim: To establish a method for cynomolgus monkey sperm cryopreservation in a chemically defined extender.Methods: Semen samples were collected by electro-ejaculation from four sexually mature male cynomolgus monkeys.The spermatozoa were frozen in straws by liquid nitrogen vapor using egg-yolk-free Tes-Tris (mTTE) synthetic extender and glycerol as cryoprotectant. The effects of glycerol concentration (1%, 3%, 5%, 10% and 15% [v/v])and its equilibration time (10 min, 30 min, 60 min and 90 min) on post-thaw spermatozoa were examined by sperm motility and sperm head membrane integrity. Results: The post-thaw motility and head membrane integrity of spermatozoa were significantly higher (P ＜ 0.05) for 5% glycerol (42.95 ± 2.55 and 50.39 ± 2.42, respectively) than those of the other groups (1%: 19.19 ± 3.22 and 24.84 ± 3.64; 3%: 34.23 ± 3.43 and 41.37 ± 3.42; 10%:15.68 ± 2.36 and 21.39 ± 3.14; 15%: 7.47 ± 1.44 and 12.90 ± 2.18). The parameters for 30 min equilibration (42.95 ± 2.55 and 50.39 ± 2.42) were better (P ＜ 0.05) than those of the other groups (10 min: 31.33 ± 3.06 and 38.98 ± 3.31; 60 min: 32.49 ± 3.86 and 40.01 ± 4.18; 90 min: 31.16 ± 3.66 and 38.30 ± 3.78). Five percent glycerol and 30 min equilibration yielded the highest post-thaw sperm motility and head membrane integrity. Conclusion:Cynomolgus monkey spermatozoa can be successfully cryopreserved in a chemically defined extender, which is related to the concentration and the equilibration time of glycerol.