In Europe, carbon dioxide therapy has been used for cardiac disease and skin problems for a long time. However there have been few reports investigating the effects of carbon dioxide therapy on skeletal muscle. Peroxisome proliferators-activated receptor (PPAR)-gamma coactivator-1 (PGC-1α) is up-regulated as a result of exercise and mediates known responses to exercise, such as mitochondrial biogenesis and muscle fiber-type switching, and neovascularization via up-regulation of vascular endothelial growth factor (VEGF). It is also known that silent mating type information regulation 2 homologs 1 (SIRT1) enhances PGC-1α-mediated muscle fiber-type switching. Previously, we demonstrated transcutaneous application of CO2 increased blood flow and a partial increase of O2 pressure in the local tissue known as the Bohr effect. In this study, we transcutaneously applied CO2 to the lower limbs of rats, and investigated the effect on the fast muscle, tibialis anterior (TA) muscle. The transcutaneous CO2 application caused: (1) the gene expression of PGC-1α, silent mating type information regulation 2 homologs 1 (SIRT1) and VEGF, and increased the number of mitochondria, as proven by real-time PCR and immunohistochemistry, (2) muscle fiber switching in the TA muscle, as proven by isolation of myosin heavy chain and ATPase staining. Our results suggest the transcutaneous application of CO2 may have therapeutic potential for muscular strength recovery resulting from disuse atrophy in post-operative patients and the elderly population.