Objective To explore the mechanism of phosphoramide mustard (PM)-induced ovarian granulosa cell damage and the effect of serum containing Zuogui pill on autophagy and apoptosis of damaged granulosa cells. Methods Serum containing Zuogui pill was prepared and primary rat ovarian granulosa cells were cultured in vitro. At 75% ~ 80% confluence, the cells were divided into four groups: group 1: control; group 2: model (M); group 3: serum containing Zuogui pill ( 10% ZGW); and group 4: model + serum containing Zuogui pill ( M + 10% ZGW). The model of chemotherapy-induced granulosa cell injury was established in groups 2 and 4 after PM treatment for 24 hours. In groups 1 and 2, 10% normal rat serum was added. In groups 3 and 4, 10% serum containing Zuogui pill was added, followed by culture for 24 hours at 37℃ with 5% CO₂ . Flow cytometry was used to detect the apoptosis rate of granulosa cells in each group and expression of Beclin-1, LC3B, P62, Bax, and Caspase-3 was detected by Western blot. Results Compared with the control group, the apoptosis rate of the model group was increased significantly, expression of Beclin-1, LC3B, Bax, and Caspase-3 protein was increased, and expression of P62 protein was decreased (P< 0. 05). The 10% serum containing Zuogui pill significantly reduced the apoptosis rate of granulosa cells in the model group, downregulated the expression of Beclin-1, LC3B, Bax, and Caspase-3 protein in damaged granulosa cells, and upregulated the expression of P62 protein in damaged granulosa cells ( P< 0. 05). Conclusions PM damages ovarian granulosa cells, promotes apoptosis of granulosa cells, and activates the autophagy / lysosome degradation pathway in granulosa cells. The 10% serum containing Zuogui pill reduces the apoptosis rate of chemotherapy-treated granulosa cells, which is related to expression of autophagy proteins.