OBJECTIVE To prepare, purify and characterize the polyclonal antibodies against CMTM4. METHODS Four polypeptides named peptide 1, 2, 3, and 4 were synthesized based on the bioinformatics analysis of the three isoforms of CMTM4, CMTM4-v1, -v2, and -v3, and coupled with keyhole limpet hemocyanin (KLH) for immunization. Among them, peptide 1, 2, and 3, common for CMTM4-v1, v2, and v3, were mixed for immunization to prepare the antibody which can recognize all of the three isoforms (Ab1); while peptide 4, which is specific for CMTM4-v1, was injected separately into New Zealand rabbits to prepare the antibody specifically targeting CMTM4-v1 (Ab2). ELISA assay was used to detect the titers of the antibodies. After purification by immunoaffinity chromatography, Ab1 and Ab2 were identified by Western blot and immunohistochemistry assays. RESULTS The titers of Ab1 and Ab2 were 1:10(5) and 1:10(6), respectively. Western blot confirmed their high specificity. Ab1 could also be used for immunohistochemistry analysis, but Ab2 could not. Immunohistochemistry analysis with Ab1 demonstrated the high expression level of CMTM4 in human testis, which was consistent with the previous result of Northern blot. Moreover, Western blot and immunohistochemistry analysis verified that Ab1 can also recognize mouse Cmtm4. CONCLUSIONS Two specific antibodies against human CMTM4 were obtained, which will be helpful for further study.