Given that the physiology of heme oxygenase-1 (HO-1) encompasses mitochondrial biogenesis, we tested the hypothesis that the HO-1 product, carbon monoxide (CO), activates mitochondrial biogenesis in skeletal muscle and enhances maximal oxygen uptake (V̇o_2max) in humans. In 10 healthy subjects, we biopsied the vastus lateralis and performed V̇o_2max tests followed by blinded randomization to air or CO breathing (1 h/day at 100 parts/million for 5 days), a contralateral muscle biopsy on day 5, and repeat V̇o_2max testing on day 8. Six independent subjects underwent CO breathing and two muscle biopsies without exercise testing. Molecular studies were performed by real-time RT-PCR, Western blot analysis, and immunochemistry. After V̇o_2max testing plus CO breathing, significant increases were found in mRNA levels for nuclear respiratory factor-1, peroxisome proliferator-activated receptor-γ coactivator-1α, mitochondrial transcription factor-A (Tfam), and DNA polymerase γ (Polγ) with no change in mitochondrial DNA (mtDNA) copy number or V̇o_2max. Levels of myosin heavy chain I and nuclear-encoded HO-1, superoxide dismutase-2, citrate synthase, mitofusin-1 and -2, and mitochondrial-encoded cytochrome oxidase subunit-I (COX-I) and ATPase-6 proteins increased significantly. None of these responses were reproduced by V̇o_2max testing alone, whereas CO alone increased Tfam and Polγ mRNA, and COX-I, ATPase-6, mitofusin-2, HO-1, and superoxide dismutase protein. These findings provide evidence linking the HO/CO response involved in mitochondrial biogenesis in rodents to skeletal muscle in humans through a set of responses involving regulation of the mtDNA transcriptosome and mitochondrial fusion proteins autonomously of changes in exercise capacity.