Vitamin E supplementation, when combined with high blood α-tocopherol (>6.25 µg/mL) at dry off, has been reported to unexpectedly increased the risk for clinical mastitis in dairy cows. Furthermore, higher levels of oxidative stress in the postpartum period were related to higher risk of mastitis. The objective of the present study was to determine the relationship between various serum biomarkers of oxidative status, incidence of mastitis, and blood α-tocopherol concentrations at dry off and at calving. A total of 146 dairy cows from a commercial farm were used in an observational field study. All cows were supplemented with 3,000 and 50 IU/cow per day of all-rac-α-tocopherol during the dry period and lactation, respectively. Blood samples were collected at dry off and at calving. Serum was analyzed for α-tocopherol, levels of reactive oxygen metabolites (ROM), thiol groups (SH), and ferric-reducing ability. Three α-tocopherol groups at calving were created: high (>3 µg/mL), medium (2-3 µg/mL), and low (<2 µg/mL). Three α-tocopherol groups at dry off were created: high (>6.25 µg/mL), medium (4.25-6.25 µg/mL), and low (<4.25 µg/mL). All cases of clinical mastitis that occurred during the dry period and the entire subsequent lactation were verified by a veterinarian. No differences were observed in the incidence of mastitis between the 3 α-tocopherol groups based on the serum levels at dry off. Incidence of mastitis was 4 times lower in the high and medium groups when compared with the corresponding value for the low-α-tocopherol group based on the serum levels at calving. Lower levels of ROM and SH at dry off and at calving were found in the group of cows with the highest α-tocopherol values at dry off when compared with the corresponding values in the low-α-tocopherol group. The ROM values at dry off but not at calving were lower in the group of cows with the highest α-tocopherol values at calving when compared with the corresponding values in the low-α-tocopherol group. No differences were observed in ferric-reducing ability values between the 3 α-tocopherol groups at dry off or calving. No differences were observed in all biomarkers of oxidative status between healthy cows and those with mastitis. Thus, blood α-tocopherol is inversely related to certain biomarkers of oxidative stress in the postpartum period and incidence of mastitis. However, reduction in the incidence of mastitis is not mediated through a reduction in the levels of various biomarkers of oxidative stress.