Equilibrium, freezing and thawing are three essential steps which play a critical role in semen freezing process. In order to optimize Horse (Equus caballus) sperm freezing methods, we investigated the impact of the equilibrium, freezing and thawing manipulations on sperm motility, membrane integrity and mitochondrial membrane potential after freeze-thawing. Semen was collected from 4 adult stallions, diluted in INRA82 + 5% clarified egg yolk + 3.5% combined cryoprotectant and then frozen. In excrement 1, we compared the effect of equilibration for 0, 45, 90, 120, 180, 240 min and 8 h on equine sperm cryopreservation; in experiment 2, we aimed to find a suitable height of liquid nitrogen steam for freezing; in exprement 3, we assessed the effect of different thawing procedures on post-thaw semen quality. Statistical analysis was conducted with ANOVA SPSS 13. Differences between means of parameters were subjected to an analysis of variance using the Tukey′s test, with P < 0.05 considered significant. The results showed that the equilibrium time of 120, 180 and 240 min resulted in significantly higher sperm motility and membrane integrity than the equilibrium for 0, 45, 90 min and 8 h group after thawing (Fig. 2); liquid nitrogen steam frozen at 2 cm and 4 cm from liquid nitrogen surface obtained similar results with program freezing method (Fig. 3); high temperature rapid thawing method (75℃ 7 s and 46℃ 20 s) gained a higher post-thaw motility than the conventional thawing method (37℃ 30 s) (Fig. 4). In summary, we believe that combination of equilibrium for 120﹣240 min, liquid nitrogen steam frozen at 2 cm and 4 cm from liquid nitrogen surface and high-temperature rapid thawing method (75℃ 7 s and 46℃ 20 s) can get better effect in horse sperm freezing process.