Mitochondria generate reactive oxygen species (ROS) under various pathophysiological conditions. In isolated mitochondria, fatty acids (FA) exhibit an uncoupling effect of the respiratory activity and modulate ROS generation. The effect of FA on intact cultured cells remains to be elucidated. The present study reports that FA (buffered by BSA) decrease the level of cellular ROS generated by the mitochondrial respiratory chain in cultured cells incubated with antimycin A. Both saturated and unsaturated FA are effective. This fatty acid-induced antioxidant effect does not result from a decrease in ROS production, but is subsequent to cellular glutathione peroxidase (GPx) activation and enhanced ROS degradation. This fatty acid-induced GPx activation is mediated through epidermal growth factor receptor (EGFR) signalling, since this response is (i) abrogated by the EGFR inhibitor AG1478 or by a defect in EGFR (in EGFR-deficient B82L fibroblasts), (ii) restored in B82LK+ cells expressing EGFR and (iii) mimicked by epidermal growth factor. These findings indicate that FA contribute to enhance cellular antioxidant defences against mitochondrial oxidative stress through EGFR-dependent GPx activation.