The effects of flax meal (FM) on the activity of antioxidant enzymes (superoxide dismutase (SOD), glutathione peroxidase (GPx) and catalase (CAT)) in the blood, mammary tissue and ruminal fluid, and oxidative stress indicators (thiobarbituric acid-reactive substances (TBARS) and 1,1-diphenyl-2-picrylhydrazyl-scavenging activity) in the milk, plasma and ruminal fluid of dairy cows were determined. The mRNA abundance of the antioxidant enzymes and oxidative stress-related genes was assessed in mammary tissue. A total of eight Holstein cows were used in a double 4 × 4 Latin square design. There were four treatments in the diet: control with no FM (CON) or 5 % FM (5FM), 10 % FM (10FM) and 15 % FM (15FM). There was an interaction between treatment and time for plasma GPx and CAT activities. Cows supplemented with FM had a linear reduction in TBARS at 2 h after feeding, and there was no treatment effect at 0, 4 and 6 h after feeding. TBARS production decreased in the milk of cows fed the 5FM and 10FM diets. There was a linear increase in nuclear factor (erythroid-derived 2)-like 2 (NFE2L2) mRNA abundance in mammary tissue with FM supplementation. A linear trend for increased mRNA abundance of the CAT gene was observed with higher concentrations of FM. The mRNA abundance of CAT, GPx1, GPx3, SOD1, SOD2, SOD3 and nuclear factor of κ light polypeptide gene enhancer in B-cells (NFKB) genes was not affected by the treatment. These findings suggest that FM supplementation can improve the oxidative status of Holstein cows as suggested by decreased TBARS production in ruminal fluid 2 h post-feeding and increased NFE2L2/nuclear factor-E2-related factor 2 (Nrf2) mRNA abundance in mammary tissue.