3-Bromopyruvate (3-BP) is a promising compound for anticancer therapy, its main mode of action being the inhibition of glycolytic enzymes, but this compound also induces oxidative stress. This study aimed at characterisation of the effect of 3-BP on the antioxidant defense system of erythrocytes. Suspensions of erythrocytes in PBS containing 5 mM glucose were treated with different concentration of 3-BP at 37°C for 1 h. Activities of antioxidant enzymes were estimated by standard colorimetric methods. The antioxidant capacity of erythrocytes was estimated using the 2,2′-azinobis(3-ethylbenzthiazoline-6-sulphonic acid) (ABTS•+) decolorisation assay and ferricyanide reduction. The content of reduced and oxidized glutathione was estimated fluorimetrically with o-phtalaldehyde. 3-BP did not affect the integrity of the erythrocyte membrane (lack of changes in the osmotic fragility). However, it induced oxidative stress in erythrocytes, as evidenced by the decrease in the content of acid-soluble thiols and reduced glutathione (GSH). Superoxide dismutase (SOD) and glutathione S-transferase (GST) activities were significantly decreased. 3-BP also decreased the transmembrane reduction of ferricyanide. Thus induction of oxidative stress in erythrocytes by 3-BP is due to depletion of glutathione and inhibition of antioxidant enzymes.