Objective: To investigate the effects of 4-Hydroperoxycyclophosphamide (4-HC), an in vitro activated metabolite of cyclophosphamide, on functional damage to the human ovarian granulosa cell line SVOG, as well as its potential mechanisms. Methods: Human ovarian granulosa cells were treated with 0.2 μM, 2 μM, and 10 μM of 4-HC for 24 h, 48 h, and 72 h, respectively. The cell viability of each group was assessed by the CCK-8 assay to determine the appropriate time and concentration for constructing an injury model. Changes in mitophagy flux were evaluated using Western blotting (WB) and real-time quantitative polymerase chain reaction (RT-qPCR). Transmission electron microscopy was used to observe mitochondrial changes in both the normal and 4-HC-injured groups. RT-qPCR was used to assess the expression of P53-related genes, while immunofluorescence was employed to detect the expression levels of P53 protein, Parkin protein, and the mitochondrial outer membrane protein TOMM20. Results: A model of in vitro injury to human ovarian granulosa cell line SVOG was successfully constructed using 2 μM 4-HC treatment for 48 h. In the 4-HC-injured SVOG cells, mitophagy flux was inhibited, and abnormal mitochondrial morphology was observed, with an increase in damaged mitochondria. P53 expression was significantly elevated in the 4-HC-injured SVOG cells. Additionally, there was an increase in cytoplasmic P53 and Parkin protein interaction, while the binding of TOMM20 and Parkin proteins was suppressed in the 4-HC-injured cells. Conclusion: In vitro, 4-HC may lead to human ovarian granulosa cell damage by inhibiting mitophagy of damaged mitochondria through the P53-Parkin pathway.