BACKGROUND: Mitochondrial dysfunction has been implicated in the pathophysiology of endometriosis, but the role of ubiquitin-specific protease 2 (USP2) remains unclear. This study aimed to determine whether USP2 modulates mitochondrial respiratory activity and proliferation in ectopic endometrial stromal cells (ESCs). METHODS: Twenty women with ovarian endometriosis who underwent laparoscopic surgery at the Department of Obstetrics and Gynecology, Xuzhou Central Hospital, between April 2024 and April 2025 were enrolled. Paired eutopic endometrial and ectopic endometriotic tissues were collected from each participant. Primary ESCs were isolated and cultured, and USP2 expression was downregulated or upregulated by lentiviral transduction. USP2 mRNA levels were quantified by reverse-transcription quantitative polymerase chain reaction, whereas the protein levels of USP2 and the mitochondrial complex IV subunits COX5B and COX6C were assessed by western blotting. Cell viability and proliferative activity were evaluated using the Cell Counting Kit-8 assay. Intracellular reactive oxygen species (ROS) levels were measured by flow cytometry, and the oxygen consumption rate (OCR) was determined using a Seahorse extracellular flux analyzer. Intracellular ATP content and mitochondrial complex IV enzymatic activity were measured using commercial assay kits. RESULTS: Compared with paired eutopic ESCs, ectopic ESCs exhibited greater proliferative activity, lower intracellular ROS levels, a higher OCR, increased intracellular ATP content, and elevated mitochondrial complex IV activity (all P < 0.01). In ectopic ESCs, USP2 knockdown reduced proliferative activity (P < 0.001), increased intracellular ROS levels (P < 0.01), and decreased OCR, intracellular ATP content, mitochondrial complex IV activity, and the protein levels of the complex IV subunits COX5B and COX6C (all P < 0.01). Conversely, USP2 overexpression in eutopic ESCs produced reciprocal changes, including increased proliferative activity, reduced intracellular ROS levels, and higher OCR, ATP content, complex IV activity, and COX5B and COX6C protein levels (all P < 0.01). Treatment with carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), a mitochondrial uncoupler, attenuated the cellular and mitochondrial changes associated with USP2 overexpression. CONCLUSIONS: USP2 was more highly expressed in ectopic than in paired eutopic endometrial tissues. Its manipulation altered ectopic ESC proliferation and mitochondrial respiratory parameters, suggesting a potential role for USP2 in the proliferative and mitochondrial phenotype of ectopic ESCs.