Background: The Chinese herbal compound Heshouwuyin has been shown to downregulate the apoptotic rate of testicular tissue cells in Wistar naturally aging rats, and this effect might be related to the mitochondrial pathway [15]. Apoptotic protease activating factor-1 (Apaf-1) is a major component of the apoptotic complex, which is a key element of the mitochondrial endogenous apoptotic pathway [13]. To further clarify the mechanism of Heshouwuyin in the mitochondrial apoptotic pathway, this study used Apaf-1 as a target to explore the mechanism by which Heshouwuyin inhibits the Apaf-1 pathway of spermatogenic cell apoptosis. Methods: In this study, an aging model of rat spermatogenic cells was established using free radical oxidative damage. Flow cytometry was used to detect the apoptosis rate of germ cells and the inhibitory effect of Heshouwuyin. Apaf-1 was specifically knocked down by siRNA interference technology, and mitochondrial membrane potential was measured. qRT-PCR, Western blotting and immunofluorescence analyses were used to detect the expression of the key genes Cyt c, Caspase-9 and Caspase-3 in the mitochondrial apoptotic pathway of spermatogenic cells. Results: Heshouwuyin reduced the mRNA and protein expression levels of Cyt c, Caspase-9 and Caspase-3 in senescent spermatogenic cells. In these cells, the mRNA and protein expression levels of Cyt c did not change significantly after specific knockdown of Apaf-1, and the mRNA and protein expression levels of Caspase-9 and Caspase-3 decreased significantly. This finding indicated that knockdown of Apaf-1 could decrease the mRNA and protein expression levels of the downstream pro-apoptotic genes Caspase-9 and Caspase-3. Although Cyt c was an upstream gene of Apaf-1, knockdown of Apaf-1 had no significant effect on Cyt c expression. Conclusion: The inhibition of spermatogenic cell apoptosis by Heshouwuyin was closely related to the Cyt c/Apaf1/Caspase-9/Caspase-3 pathway. The inhibition of apoptosis by Heshouwuyin not only involved the Apaf-1 pathway, but other signaling pathways.