Objective：To investigate the effect of the in vitro activation product of cyclophosphamide，4-hydroperoxy cyclophosphamide（4-HC），on the functional impairment of the human ovarian granulosa cell line SVOG，and the potential underlying mechanisms. Methods：SVOG cells were treated with 0.2，2.0，and 10.0 μmol/L of 4-HC for 24，48，and 72 h. The cell viability in each group was measured using the CCK-8 assay to determine the optimal time and concentration for constructing an injury model. Western blot and RT -qPCR were used to detect changes in mitochondrial autophagy flux. Transmission electron microscopy was employed to observe mitochondrial changes in both normal and 4-HC -injured cells. RT -qPCR was used to assess the expression of P53- related genes，and immunofluorescence was applied to detect the expression levels of P53，Parkin，and the translocase of the outer mitochondrial membrane 20（TOMM20）proteins. Results：A model of SVOG cell injury induced by 2.0 μmol/L 4-HC for 48 h was established in vitro. Mitochondrial autophagy flux was inhibited，and mitochondrial morphology was abnormal in 4-HC-injured SVOG cells，with a significant increase in damaged mitochondria. The expression level of P53 was significantly increased in 4-HC -injured SVOG cells. An increase in the cytoplasmic interaction between P53 and Parkin protein was observed，while the binding of TOMM20 and Parkin protein was inhibited in 4-HC -injured SVOG cells. Conclusion：In vitro，4-HC may induce damage to human ovarian granulosa cells by inhibiting mitochondrial autophagy through the P53-Parkin pathway.