Abstract: Objective Diminished ovarian reserve (DOR) is a major cause of female fertility decline, yet its underlying mechanisms remain incompletely elucidated. Apurinic/apyrimidinic endonuclease 1 (APE1) and DNA polymerase beta (Polβ) are key enzymes of the base excision repair (BER) pathway. This study aimed to investigate the expression alteration of these two genes in the granulosa cells of patients with DOR and their associations with DNA damage, mitochondrial dysfunction, and cellular apoptosis. Methods This was retrospective study A total of 168 young women (age ≤ 35 years) undergoing assisted reproductive technology (ART) treatment at the Reproductive Medicine Center of the First Hospital of Lanzhou University were enrolled in this study. Participants were stratified by ovarian reserve status into a DOR group and a normal ovarian reserve (NOR) group (n = 84 per group). Ovarian granulosa cells were isolated by density gradient centrifugation. Real-time quantitative PCR (RT-qPCR), fluorescence staining, immunocytochemistry, and flow cytometry were employed to assess oxidative stress and DNA damage markers, mitochondrial functional status, transcriptional alterations of mitochondrial DNA (mtDNA) maintenance-related genes, the occurrence of apoptosis, and functional alterations of the base excision repair (BER) pathway, including the protein expression levels of APE1 and Polβ. Correlation analyses were subsequently performed to evaluate the associations among the aforementioned indicators. Results Compared with the NOR group, granulosa cells from the DOR group exhibited significantly elevated oxidative stress, as evidenced by increased levels of reactive oxygen species (ROS) and malondialdehyde (MDA), upregulated mRNA expression of heme oxygenase-1 (HMOX-1), and downregulated mRNA expression of superoxide dismutase 2 (SOD2) (all p < 0.01). Protein expression levels of the BER pathway enzymes APE1 and Polβ were markedly reduced, while the nuclear DNA damage marker γH2AX was elevated. The mtDNA damage response gene ribonucleotide reductase regulatory TP53- inducible subunit M2B (RRM2B) was upregulated, mitochondrial membrane potential was decreased, and the apoptosis rate was significantly increased. Correlation analyses revealed that APE1 expression levels were negatively correlated with γH2AX (r=-0.6758, p=0.0158), Polβ expression levels were positively correlated withmitochondrial membrane potential (r=0.7005, p=0.0112), and γH2AX levels were positively correlated with the apoptosis rate (r=0.7492, p=0.0050). Conclusion Granulosa cells from patients with DOR exhibited impaired BER capacity, elevated reactive oxygen species (ROS) levels, nuclear DNA and mtDNA damage, mitochondrial dysfunction, and increased apoptotic rates. These findings suggest that the aforementioned pathological alterations may collectively contribute to the onset and progression of DOR. Furthermore, APE1 and Polβ may serve as candidate biomarkers for evaluating DNA repair capacity in ovarian granulosa cells.