We report the effect of cross-sex hormonal replacement on antioxidant enzymes from rat retroperitoneal fat adipocytes. Eight rats of each gender were assigned to each of the following groups: control groups were intact female or male (F and M, resp.). Experimental groups were ovariectomized F (OvxF), castrated M (CasM), OvxF plus testosterone (OvxF + T), and CasM plus estradiol (CasM + E2) groups. After sacrifice, retroperitoneal fat was dissected and processed for histology. Adipocytes were isolated and the following enzymatic activities were determined: Cu-Zn superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST), and glutathione reductase (GR). Also, glutathione (GSH) and lipid peroxidation (LPO) were measured. In OvxF, retroperitoneal fat increased and adipocytes were enlarged, while in CasM rats a decrease in retroperitoneal fat and small adipocytes are observed. The cross-sex hormonal replacement in F rats was associated with larger adipocytes and a further decreased activity of Cu-Zn SOD, CAT, GPx, GST, GR, and GSH, in addition to an increase in LPO. CasM + E2 exhibited the opposite effects showing further activation antioxidant enzymes and decreases in LPO. In conclusion, E2 deficiency favors an increase in retroperitoneal fat and large adipocytes. Cross-sex hormonal replacement in F rats aggravates the condition by inhibiting antioxidant enzymes.