Abstract: Objective To investigate the expression and clinical significance of paired box gene 6 (PAX6) and microRNA-375-3p (miR-375-3p) in ovarian endometriosis (EMs). Methods A total of 40 patients who were admitted to the Affiliated Hospital of Xuzhou Medical University from December 2022 to August 2024 and laparoscopically diagnosed with EMs were selected. Their wall tissues of ovarian endometrioma were collected as an ectopic group, while the eutopic endometrial tissues were collected as an eutopic group. Additionally, normal endometrial tissues were collected from 40 patients who underwent hysterectomy for uterine fibroids and set as a control group. The expression of PAX6 protein was analyzed by immunohistochemistry. The levels of PAX6 mRNA and miR-375-3p in the tissues were detected by real-time fluorescence quantitative PCR (qRT-PCR). The relationships between PAX6 mRNA, miR-375-3p, and the clinical pathological features of ovarian EMs were analyzed, and the correlation between PAX6 mRNA and miR-375-3p was assessed. Receiver operating characteristic (ROC) curves were plotted to evaluate the diagnostic value of PAX6 mRNA and miR-375-3p for ovarian EMs. Results Compared with the control group, patients with ovarian EMs showed increased levels of PAX6 protein and mRNA, and decreased miR-375-3p levels (P<0.05). Compared with the eutopic group, increased PAX6 protein and mRNA expression and decreased miR-375-3p expression were observed in the ectopic group (P<0.05). In ovarian EMs patients with high CA125 levels, their PAX6 mRNA positive rate increased but miR-375-3p positive rate decreased, with statistical differences (P<0.001). No statistical differences were found in PAX6 mRNA and miR-375-3p expression among ovarian EMs patients with different ages, lesion sizes, clinical stages, or degrees of dysmenorrhea (P>0.05). A negative correlation was found between PAX6 mRNA and miR-375-3p expression in ovarian EMs patients (r=-0.522, P<0.05). According to ROC curve, the area under the curve (AUC) for PAX6 mRNA, miR-375-3p, and their combination for predicting ovarian EMs was 0.943, 0.846, and 0.953, respectively, with better diagnostic value in the combined diagnosis than PAX6 mRNA or miR-375-3p alone. Conclusions PAX6 and miR-375-3p are abnormally expressed in ovarian EMs. Their combination may provide new directions for the diagnosis and treatment of ovarian EMs.