Prochloraz is a widely used fungicide in the world, which belongs to the environmental endocrine disrupting estrogen pesticide of reproductive system. The purpose of the present study was to investigate Prochloraz on reproductive system of male offspring mice after perinatal exposure to by prochloraz. The different doses of Prochloraz (group MA, MB and MD) were given to male father mice by intragastric administration. The results firstly showed that the average testicular weight in different dosage groups were significantly lower than the control group (0.312g ,0.294g, Vs 0.355g; P < 0.05), and the testicular coefficient index in group MB and MD is also lower than the control group. Secondly, all valid RNA-Seq data were successfully mapped to the mouse genome database for further analysis. Thirdly, we found that significantly expressed mRNAs clustered in group B and D compared to control. Finally, the results showed that male mouse reproductive relative genes response to prochloraz invasion were greatly changed, which involved in the regulation of sperm mitochondria function by two genes (Mt-nd6 and Slc12a4) , and six genes (Greb1, Esrrb , Catsperb, Mospd2, Sohlh1 and Specc1) is closely related to sperm functions and estrogen reaction. It can be concluded that prochloraz play an important role on male offspring reproductive system, which lays a foundation for the follow-up evaluation of its reproductive toxicological effects.