In the present study, by using a serologic proteomic analysis, we identified phosphoglycerate mutase isozyme B (PGAM-B) as a putative target of autoantibodies in autoimmune hepatitis (AIH). To evaluate whether the identified autoantigen is crucial for AIH, we cloned PGAM-B cDNA and expressed the recombinant protein in Escherichia coli. The soluble PGAM-B was purified by affinity chromatography and used as a coating antigen to determine the frequency of the PGAM-B-autoantibodies (PGAM-B-Abs) in patients with AIH and primary biliary cirrhosis (PBC) as well as chronic hepatitis B (CHB), chronic hepatitis C (CHC), and healthy donors by ELISA. Our study showed that the autoantibody to PGAM-B was predominantly present in AIH patients and 70.04% (50/71) of the tested AIH sera reacted to PGAM-B. The frequency of autoantibodies to PGAM-B is much higher in patients with AIH than in patients with PBC, CHB, CHC, and normal control. The data were further confirmed by using 1-DE Western blot analysis. Our study presents the first description of this protein as a candidate of diagnostic marker for AIH.