To date, there is no information available concerning the relationship between administration of CLA and changes in oxidative status of bovine mammary gland. The aim of this study was to assess in vitro the role of CLA in cell protection against the oxidative stress of bovine mammary cells. The uptake rates of CLA in BME-UV1 cells at 3 h and 48 h were tested using DAD HPLC analysis. BME-UV1 cells were treated with complete medium containing 50 µM of cis-9,trans-11 CLA (c9,t11), trans-10,cis-12 CLA (t10,c12) and CLA Mixture (50% c9,t11 and 50% t10,c12). After 48 h from addition of CLA, cell samples were collected for determining oxidative markers such as nicotinamide adenine dinucleotide phosphate (NADPH), glutathione (GSH/GSSG), thiobarbituric acid reactive substances (TBARS), cytoplasmic superoxide dismutase (SOD), glutathione peroxidase (GPx1), glutathione S-transferase (GST) and glutathione reductase (GR) using commercial kits. The mRNAs quantification of antioxidant enzymes (SOD, GPx1, GST and GR) was performed by rt-PCR. The potential protection of CLA against H₂O₂-induced oxidative stress was assessed by XTT assay after 48h of pre-treatment with CLA and 3h with H₂O₂. The data were analyzed by ANOVA and differences were declared significant at P < 0.05. The results showed that the uptake rates of CLA in cells increased from 3 (26.54%) to 48h (73.95%). CLA increased (P < 0.01) the concentration of reduced GSH (CLA 17.25 vs control 11.73 µM) and NADPH (CLA 0.28 vs control 0.20 µM), mostly in cells treated with t10,c12 (0.50 µM). The enzymatic antioxidant activity of GR (CLA 7.00 vs control 11.82 U/L) was decreased (P < 0.01) whereas GSPx1(P < 0.01), GST (P < 0.05) and SOD (P < 0.01) activities (CLA 19.85, 0.70, 0.05 vs control 7.07 U/L, 0.61 U/mL, 0.02 U/mL, respectively) were increased in cells treated with CLA. Lower levels of TBARS (CLA 1.50 vs control 1.96 µM/MDA equivalent) were observed in cells treated with CLA. CLA had not any substantial effect on gene expression of antioxidant enzymes. All CLA isomers were able to enhance (P < 0.01) cell resistance against oxidative stress (CLA 2.85 vs control 2.65 O.D.). Findings of the present study corroborate the antioxidant role of CLA, in particular of t10,c12 isomer, by improving of redox status in cells, and might be of help during physiological oxidative stress situations such as the periparturient period in dairy cow. This work was funded by BASF-SE.