Here, we found that formate, an essential one-carbon metabolite, activates superoxide (O2·-)/hydrogen peroxide (H O ) release from mitochondria. Sodium formate (30 μm) induces a significant increase in O2·-/H O production in liver mitochondria metabolizing pyruvate (50 μm). At concentrations deemed to be toxic, formate does not increase O2·-/H O production further. It was observed that the formate-mediated increase in O2·-/H O production is not associated with cytochrome c oxidase (COX) inhibition or changes in membrane potential and NAD(P)H levels. Sodium formate supplementation increases phosphorylating respiration without altering proton leaks. Finally, it was observed that the 2-oxoglutarate dehydrogenase (OGDH) inhibitors 3-methyl-2-oxovaleric acid (KMV) and CPI-613 inhibit the formate-induced increase in pyruvate-driven ROS production. The importance of these findings in one-carbon metabolism and physiology are discussed herein.