Myasthenia gravis (MG) is considered as an autoimmune disease mainly mediated by antibodies against acetylcholine receptor. In recent years, other targets related to MG have been the subject of interest. Our previous research found that protein P25 was lower in muscles of MG patients using two-dimensional electrophoresis. In present study, anti-serum to P25 was prepared, immunohistochemistry and ATPase staining revealed that P25 was a muscle specific cytosolic protein and was mainly distributed in type I muscle fibers. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and precise molecular weight derived from mass spectrometer identified P25 as carbonic anhydrase III (CA III). Some members of CA family are related to autoimmune diseases and CA III is recently reported to be involved in rheumatoid arthritis. The results of immunoblot in this report showed that the level of CA III is specifically insufficient in the skeletal muscle of MG patients. The possible roles that CA III play in MG need further elucidation.