Objective: As female reproductive cells, the quality of oocytes after fertilization plays a crucial role in embryonic development. This study investigated the effect and mechanism of 4-hydroxycyclophosphamide (4-HC), the active metabolite of cyclophosphamide (CTX), on oocyte quality. Methods: Mouse cumulus oocyte complexes (COCs) in the germinal vesicle (GV) stage were randomly divided into eight groups. The blank control group received no treatment, while the solvent control group was treated with DMSO at the same concentration as the experimental group. The experimental groups were exposed to 4-HC at concentrations of 0.3 μM, 1 μM, 3 μM, 10 μM, 30 μM, and 100 μM. The in vitro matured oocytes were observed for the first polar body discharge rate, 2-cell rate, and blastocyst rate. The 1 μM concentration group was further evaluated for reactive oxygen species (ROS) levels, mitochondrial membrane potential (MMP), and reduced glutathione (GSH) content to assess oocyte quality and explore the mechanism. QPCR and immunofluorescence were used to detect the effect of 4-HC on DNMT3A expression in oocytes. Results: With increasing 4-HC concentration, the 2-cell rate of oocytes remained similar or slightly decreased, while the blastocyst rate decreased significantly (P < 0.05). Treatment with 1 μM 4-HC reduced mitochondrial membrane potential (P < 0.05), increased intracellular superoxide anion content (P < 0.05), and decreased reduced glutathione content (P < 0.05). Additionally, and the blastocyst formation rate (0.8094 ± 0.05014 vs. 0.5657 ± 0.1010, P < 0.05) were significantly reduced. PCR and immunofluorescence results showed increased DNMT3A expression in oocytes (P < 0.05). Conclusion: 4-HC induces oxidative stress and mitochondrial damage in oocytes, impairing their developmental potential and affecting oocyte epigenetics.