The process of cryopreservation results in high concentration of reactive oxygen species which is detrimental to spermatozoa. The aim of this study was to investigate whether addition of vitamin E analogue to freezing extender can facilitate the cryosurvival of spermatozoa in rabbits, and how vitamin E protects spermatozoa against damages during the process of preservation. Freshly ejaculated semen was diluted with Tris-citrate-glucose extender supplemented with different concentrations of Trolox (a vitamin E analogue). The level of radical oxygen species (ROS) in spermatozoa that was exposed to Trolox was significantly lower than that of the control during each step of the process of preservation. The percentage of frozen-thawed spermatozoa with lipid peroxidation in the Trolox treatments was significantly lower than that of the control. The motility, intact acrosome, membrane integrity and mitochondrial potentials of the frozen-thawed spermatozoa in the treatment of 200 μM Trolox were significantly higher than those of the control. These observations suggest that addition of vitamin E to a freezing extender leads to higher integrity of acrosome, plasma membrane and mitochondrial membrane potential as well as higher motility. Vitamin E protects spermatozoa through its capacity to quench ROS accumulation and lipid peroxidation during the process of preservation. Addition of Trolox is recommended to facilitate the improvement of semen preservation for the rabbit breeding industry.