Objective To investigate the mechanism of Zuogui Pills (ZGP) on improving the stemness of ovarian germline stem cells (OSCs) in ovarian aging rats through the gap junction protein connexin 43 (Cx43) phosphorylation based on the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) pathway. Methods Eight-week-old female SD rats were randomly divided into control(CON),model(cyclophosphamide,CTX),ZGP and dehydroepiandrosterone(DHEA) groups,six rats in each group. CTX was injected intraperitoneally for 15 days to establish a model for ovarian aging,and then ZGP (1.85 g·kg-1) or DHEA (8.1 mg·kg-1) was administered by gavage once daily for 30 days. Follicle count at different stages and pathological changes of ovarian tissues were observed by HE staining. ELISA was used to detect serum levels of anti-mullerian hormone (AMH),follicle-stimulating hormone (FSH),and estradiol(E2). The expression levels of related proteins (PCNA,MVH,Oct4,Cx43,p-Cx43,EGFR,AKT、p-AKT) in ovarian tissue were detected by Western Blot. Results Compared with the CON group,rats in CTX group had significantly reduced body mass(P<0.05) and ovarian mass(P<0.05). The number of primordial follicles decreased significantly(P<0.01). Serum FSH level increased remarkably(P<0.01),while AMH and E2 levels showed a downward trend (P>0.05). The protein expressions of Oct4,MVH,PCNA,EGFR,p-AKT/AKT,p-Cx43(Ser368)/Cx43 and Cx43 in ovarian tissue were obviously decreased(P<0.05,P<0.01). Compared with the model group,the body mass of rats in ZGP group was significantly increased (P<0.05),as well as ovarian mass and ovarian coefficient were remarkably increased (P<0.01). The number of primordial follicles were increased significantly (P<0.01),and E2 level increased significantly (P<0.05). The protein expressions of Oct4,MVH,PCNA,EGFR,p-AKT/AKT,p-Cx43(Ser368)/Cx43 and Cx43 in ovarian tissue were all increased significantly (P<0.05,P<0.01). Conclusion ZGP may ameliorate ovarian aging by maintaining the stemness of OSCs. Its mechanism of actions is related to the PI3K/AKT signaling pathway and gap junction protein phosphorylation.