The aim of the study was to evaluate glutathione redox state, glutathione peroxidase (GPx) activity, and selenium (Se), non-esterified fatty acids (NEFA) and β-hydroxybutyrate (BHB) concentrations in 15 Holstein periparturient dairy cows and to monitor the effect of negative energy balance (NEB) on the level of oxidative processes ongoing in dairy cows in postpartum period. The body condition score (BCS) was recorded and blood samples were collected 4 times during periparturient period. A significantly increased NEFA concentration was recorded on calving day (P < 0.05) and 7 days post partum (p.p.; P < 0.01) compared to 7 days ante partum (a.p.). The reduced glutathione (GSH) concentration was significantly decreased on calving day and 7 days p.p. (P < 0.05) as compared to 7 days a.p. The oxidized glutathione (GSSG) concentration was significantly higher 7 days p.p. as compared to calving day (P < 0.01) and 14 days p.p. (P < 0.05). Between the GSSG concentration and the GSH/GSSG ratio was found a significantly negative (r = −0.84; P < 0.001) correlation. The significant decrease in GPx activity was found 14 days p.p. as compared to 7 days a.p. (P < 0.05). The BCS value was significantly positively correlated (r = 0.44; P < 0.05) with GSSG concentration. The results of the study indicate significant changes of antioxidant/oxidant markers and also confirm that in the postpartum period oxidative stress occurs in dairy cows. It also seems that BCS correlates with these indicators and may influence the level of oxidative processes in cows during the periparturient period.