We hypothesized that in addition to serving as a fuel source and gluconeogenic precursor, lactate anion (La⁻) is a signaling molecule. Therefore, we screened genome‐wide responses of L6 cells to elevated (10 and 20 mM) sodium‐La⁻ added to buffered, high‐glucose media. Lactate increased reactive oxygen species (ROS) production and up‐regulated 673 genes, many known to be responsive to ROS and Ca²⁺. The induction of genes encoding for components of the mitochondrial lactate oxidation complex was confirmed by independent methods (PCR and EMSA). Specifically, lactate increased monocarboxy‐late transporter‐1 (MCT1) mRNA and protein expression within 1 h and cytochrome c oxidase (COX) mRNA and protein expression in 6 h. Increases in COX coincided with increases in peroxisome prolif‐erator activated‐receptor γ coactivator‐1α (PGCla) expression and the DNA binding activity of nuclear respiratory factor (NRF)‐2. We conclude that the lactate signaling cascade involves ROS production and converges on transcription factors affecting mi‐tochondrial biogenesis.—Hashimoto T., Hussien, R., Oommen, S., Gohil, K., Brooks G. A. Lactate sensitive transcription factor network in L6 cells: activation of MCT1 and mitochondrial biogenesis. FASEB J. 21, 2602–2612 (2007)