Objective：To discuss the effects of gene silencing of sperm β-actin on the vitality of sperm and pregnant rate of rats. Methods：Firstly，lentivirus carrying shRNA sequence was constructed to down regulate the expression of ACTB gene. Secondly，the 60 male and 60 female rats were randomly divided into three groups：blank control group（normal saline group），empty lentivirus group（5X-1- RNAi-LV group） and experimental group（ACTB- RNAi-LV group）. Male rats were administrated with saline or lentivirus solution by tissue injection at testis at the fourth week. Rats were mated when they reached sexual maturity. Finally，the pregnancy rate of female rats and the motility of sperm were observed. The expression of ACTB gene and β-actin in sperm were detected by reverse transcription PCR and Western blot. Results：The model of sperm expressing low level of β-actin was successfully construed. The expression of β-actin and mRNA of ACTB gene as well as pregnant rate in experimental group were significantly reduced compared with those in blank control group and empty lentivirus group（P<0.05）.There was no difference in the expression of β-actin and pregnant rate in blank control group and empty lentivirus group（P >0.05）. Conclusion：The motility of sperm expressing the low level of β-actin could decline，which leads to the fall of pregnant rate.