Objective:To explore the potential protective effects and mechanism of isocitric acid in post-thawing sperm. Methods:After routine semen analysis,semen samples from 16 fertile males were selected. Every sample was equally divided into five groups. One group was mixed with routine protectant as negative control,and the other groups were mixed with modified protectant containing different concentration of isocitric acid. After and before thawing,the sperm samples were subjected to CASA for analysis and flow cytometry for mitochondrial transmembrane potential,respectively. Results:The percentage of forward movement sperm in G2 and G3 group was higher than that of the control group(P < 0.05 and P < 0.01). Sperm concentration of G3 group also increased(P < 0.05). The mitochondrial membrane potential in the G3 group was higher than that of the control group (P < 0.05).Conclusion:Adding a certain concentration of citric acid in the cryoprotectant can reduce the damage of the mitochondria during the freezing and thawing.