Cancer/testis (CT) antigens are promising targets for immunotherapy due to their restricted expression in the germ cells of the testis in healthy tissue and high immunogenicity. The aim of the present study was to determine whether transmembrane protein 31 (TMEM31) is a CT antigen and to investigate the pattern of TMEM31 expression during the progression of melanoma. The pattern of expression of human TMEM31 mRNA in multiple human tissues was determined through reverse transcription-polymerase chain reaction analysis. TMEM31 protein expression was analyzed in the human testis, in addition to 128 primary melanoma and 64 metastatic melanoma samples through immunohistochemistry analysis. TMEM31 was identified to be predominantly expressed in the testis and weakly expressed in the placenta. In addition, TMEM31 protein expression was detected in 120/190 (63.16%) melanoma samples (primary and metastatic). The intensity of TMEM31 staining in metastatic and primary melanomas was determined through semiquantitative integrated optical density (IOD) analysis, and identified to be significantly increased in metastatic melanoma compared with primary melanoma (0.24 +/- 0.03 vs. 0.09 +/- 0.01 IOD/area; P<0.001). The expression of TMEM31 protein was significantly increased in metastatic compared with primary melanoma samples (76.56 vs. 56.35%; P=0.017). The results of the present study suggest that TMEM31 is a novel CT antigen that serves an essential role in melanoma metastasis, in addition to being a potential immunotherapeutic target for the treatment of patients with melanoma.