Antioxidant enzymes seem to play roles in controlling the luteal function and the luteolytic action of prostaglandin F2α (PGF). The aim of this study was to clarify the roles of catalase (CAT) and glutathione peroxidase (GPx) in the luteolytic action of PGF in both corpus luteum (CL) and cultured luteal cells. Corpora lutea were collected at the early (days 2-3), developing (days 5-6), mid (days 8-12), late (days 15-17) and regressed (days 19-21) luteal stages (n = 5 CL/stage) and at 0, 2 and 24 h after luteolytic PGF administration (0 h) on day 10 (n = 5 cows/time point). Catalase protein and the activities of CAT and GPx increased from the early to mid-luteal stage, then all decreased (P < 0.05), reaching their lowest levels at the regressed luteal stage. The levels of GPx1 protein were lower in the regressed luteal stage than in other stages (P < 0.05). Immunohistochemical examination also revealed the expression of CAT and GPx1 protein in the bovine CL tissue. Injection of a luteolytic dose of PGF increased luteal GPx1 protein and GPx activities at 2 h but suppressed them at 24 h. Catalase protein and CAT activity did not change at 2 h but CAT activity decreased (P < 0.05) at 24 h. Prostaglandin F2α (1 µM) and H2O2 (10 µM) decreased CAT and GPx1 protein expression and activity at 24 h in cultured luteal cells isolated from mid-luteal stage CL (n = 3 CL per each experiment). Interestingly, CAT protein and activity did not change while GPx1 protein and activity increased at 2 h in luteal cells treated with PGF and H2O2 (P < 0.05). The down-regulation of CAT and GPx, and their activities during structural luteolysis might enhance the accumulation of reactive oxygen species, which would result in both increasing luteal PGF production and cell death to complete CL regression in cattle.