Mitochondrial ATP production can be impaired by oxidative stress. Glutathione peroxidase 4 (Gpx4) is an antioxidant defense enzyme found in mitochondria as well as other subcellular organelles that directly detoxifies membrane lipid hydroperoxides. To determine if Gpx4 protects ATP production in vivo, we compared mitochondrial ATP production between wild-type mice and Gpx4 transgenic mice using a diquat model. Diquat (50 mg/kg) significantly decreased mitochondrial ATP synthesis in livers of wild-type mice; however, no decrease in mitochondrial ATP synthesis was detected in Gpx4 transgenic mice after diquat. We observed no differences in activities of mitochondrial respiratory chain complexes between Gpx4 transgenic mice and wild-type mice. However, compared to wild-type mice, diquat-induced loss of mitochondrial membrane potential was attenuated in Gpx4 transgenic mice. Therefore, our results indicate that decreased ATP production under oxidative stress is primarily due to reduced mitochondrial membrane potential and overexpression of Gpx4 maintains mitochondrial membrane potential under oxidative stress.