Objective To investigate the mechanism of Heshouwuyin in anti-aging in testis of the aging model rats by detecting expression of Rb,p16, p53, p19 and p21 in aging rats testis. Methods Totally 84 of 8 month male SD rats were selected and randomly divided into normal group, model group, Heshouwuyin high，middle，low group, Heshouwuwan group and spontaneous recovery group with 12 rats in each group The sub-acute aging rats were made by injecting D-gal into abdominal cavity continually. SP immunohistochemistry, Western blotting and RT-PCR were used to observe the differential expression of phosphorylated Rb protein, p16, p53, p19 and p21 in testis tissue. Results The expression of testis tissue Rb, p53, p16, p19 and p21 in model group rat obviously increased, the expression of pRb obviously decreased (P<0.05); Heshouwuyin and Heshouwuwan group raised the expression of pRb, decreased the expression of Rb, p53, p16, p19, p21(EM>P/EM><0.05), and the regulating effect of Heshouwuyin high group was more effective than rest groups(EM>P/EM><0.05). Conclusions Heshouwuyin regulates the expression of Rb/p53 signal transduction pathway related gene and protein in testis tissue to