Cancer/testis (CT) antigens exhibit highly tissue-restricted expression and are considered promising targets for cancer vaccines. Here we identified a novel CT gene ZNF645 which restrictively expresses in normal human testes and lung cancer patients (68.3%). To investigate the promoter methylation status of ZNF645, we carried out bisulfite genomic sequencing and found that the CpG island in its promoter was heavily methylated in normal lung tissues without the expression of ZNF645, whereas there was high demethylation in normal human testes and lung carcinoma tissues with its expression. Also ZNF645 could be remarkably activated in A549 and HEK293T cells treated by DNA demethylation agent 5'-aza-2'-deoxycytidine. And the dual luciferase assay revealed that the promoter activity of the ZNF645 was inhibited by methylation of the CpG island region. Therefore, we proposed that ZNF645 is a CT gene and activated in human testis and lung cancers by demethylation of its promoter region.