Cis-aconitic acid decarboxylase (CAD) has been assumed to be a key enzyme in the production of itaconic acid. Here we aimed to efficiently generate the monoclonal antibody against the CAD protein. We synthesized the peptide "pdnaelvlltlgqawqg" based on the published CAD cDNA sequences. The peptide was chemically linked with the carrier protein keyhole limpet hemocyanin and then injected into Balb/c mice. Hybridomas were screened by indirect enzyme-linked immunosorbent assay (ELISA) using either purified 6 x His-CAD fusion protein or the peptide. One MAb named K2 (IgG1), effective in detecting the native CAD protein, was characterized by ELISA and Western immunoblotting. By using the MAb, we found that the CAD protein was more highly expressed in poorly differentiated gastric cancer tissues than in well-differentiated and moderately differentiated tissues. Taken together, the MAb K2 would be helpful for understanding the functions of CAD in gastric carcinogenesis.