Abstract: Objective: This study aimed to investigate the lipid metabolism characteristics, particularly lipid uptake and accumulation, of regulatory CD4+ T cells (Tregs) in the ovarian cancer microenvironment, and its potential impact on their PD-1/CTLA-4. Methods: The lipophilic fluorescent dye BODIPY? 493/503 and the fluorescent fatty acid probe BODIPY? 500/510 C1 C12 were used to detect intracellular lipid content and lipid uptake capacity, respectively, in human CD4+Tregs isolated from ovarian cancer tissues or co-cultured with supernatants from various ovarian cancer cell lines (ES-2, SKOV3, CAOV3). Lipid metabolism was modulated using the fatty acid oxidation inhibitor Etomoxir, the fatty acid synthesis inhibitor C75, and the fatty acid uptake inhibitor Sulfo-N-succinimidyl oleate (SSO). Lipid content and the expression of immunosuppressive molecules PD-1 and CTLA-4 were analyzed by flow cytometry. Results: CD4+Tregs infiltrating ovarian cancer tissues exhibited significantly higher lipid content and lipid uptake capacity compared to conventional CD4+T cells (Tconv) (P < 0.01). Among the ovarian cancer TSNs tested in vitro, CAOV3-derived TSNs most significantly enhanced intracellular lipid content and uptake capacity in CD4+Tregs relative to basal medium (P < 0.05). Furthermore, CAOV3-conditioned medium upregulated PD-1 and CTLA-4 expression in CD4+Tregs (P < 0.05). This was accompanied by a concentration-dependent increase in both lipid accumulation and fluorescent fatty acid analog uptake (P < 0.05). Notably, the fatty acid uptake inhibitor SSO effectively reversed the CAOV3 supernatant-induced lipid accumulation (P < 0.05) in CD4?Tregs and the elevated expression of PD-1 (P < 0.01) and CTLA-4 (P < 0.05), but not by the oxidation inhibitor Etomoxir or the synthesis inhibitor C75. Conclusion: The ovarian cancer microenvironment promotes lipid uptake in CD4+Tregs, leading to intracellular lipid droplet accumulation, which in turn enhances their immunosuppressive function, as evidenced by upregulated PD-1 and CTLA-4 expression. Targeting the fatty acid uptake pathway may represent a potential strategy to reverse Treg-mediated immunosuppression in ovarian cancer. Key words: ovarian cancer; CD4+Tregs; Lipid; PD-1/CTLA-4