We aimed to establish the first monoclonal antibody (MAb) against the human EMILIN-5 protein (elastin microfibril interface located protein-5) and to investigate its distribution in normal human esophageal tissues and esophageal carcinomas. The bacterially expressed 6 His-EMILIN-5 fusion protein was induced and purified. Hybridomas were screened by indirect enzyme-linked immunosorbent assay (ELISA) using either purified 6x His-EMILIN-5 fusion proteins or purified 6x His-ZNRD1 fusion protein as a control. The EMILIN-5 protein-specific MAb was further identified by Western immunoblot analysis. The expression of EMILIN-5 was investigated in 63 cases of esophageal cancer specimens and 60 cases of normal esophageal specimens using immunohistochemical analysis. The expression of 6x His-EMILIN-5 fusion proteins was successfully induced. One MAb, H7 (IgG1), effective in detecting the recombinant and the cellular protein, was characterized. H7 bound to native EMILIN-5 protein and should be useful in studies of EMILIN-5 protein function and expression. EMILIN-5 staining was found positive in 37 cases of esophageal cancer tissues (59%) and 7 cases of normal esophageal tissues (12%). The higher-grade frequency of expression of EMILIN-5 in normal esophageal tissues was significantly lower (p < 0.01) than that in tumor tissues. We concluded that EMILIN-5 might play important roles in carcinogenesis of esophageal cancer.