Objective To investigate whether Heshouwuyin can delay the aging of Leydig cells in rat testis through DNA methyltransferase 1 (DNMT1). Methods  Totally 40 male Wistar rats were randomly divided into 4 groups, with 10 rats in each group. Immunohistochemistry was used to detect the expression levels of DNMT1 in testis tissue of rats. Testosterone content in serum of rats in each group was detected by ELISA test. A rat Leydig cell aging model was established by free radical oxidative damage. DNMT1 was knocked down by lentivirus in Leydig cells, and the cell senescence status and the testosterone content and testosterone synthesis key enzyme 3β-hydroxysteroid dehydrogenase (3β-HSD), cytochrome P450 family member 11A1 (CYP11A1) content secreted by cells were detected by β-GALactosidase（β-GAL）staining, immunofluorescenct staining and ELISA. Results   Compared with the young control group (YCG), the expression of P16 protein and the positive rate of β-GAL in the testis tissue of rats in the natural aging group (NAG) increased significantly, and the expression of DNMT1 and serum testosterone levels decreased(P<0.05). However, after Heshouwuyin intervention, the expression of P16 protein and the positive rate of β-GAL in the testis of aging rats were reduced, and DNMT1 expression and the serum testosterone levels increased (P<0.05). The same trend was observed in Leydig cells. Knockdown of DNMT1 in Leydig cells, β-GAL positivity and P16 protein expression increased significantly, and testosterone secretion and testosterone synthesis key enzymes 3β-HSD, CYP11A1 content from Leydig cells decreased significantly, compared with the normal control group (NCG) (P<0.05). When Heshouwuyin was added, the above phenomenon was improved. Conclusion Heshouwuyin can delay the aging of rat Leydig cells through DNMT1.