TMEPAI (transmembrane prostate androgen-induced protein) has been reported to be overexpressed during tumour progression; however, little is known concerning transcriptional mechanisms regulating TMEPAI gene expression. In this study, the TMEPAI gene promoter has been identified and characterized, and the effects of Sp1 on TMEPAI-induced viability of A549 cells were evaluated, using MTT and colony formation assays. We found that the sequence between -298 and +24 consists of basal promoter activity for TMEPAI. Further analysis indicated that two Sp1-binding sites are crucial for maintaining basal transcriptional activity of the TMEPAI promoter, and chromatin immunoprecipitation assays confirmed direct binding of Sp1 to the TMEPAI promoter. In addition, Sp1 up-regulated TMEPAI protein expression, as well as Sp1 promoting TMEPAI-induced cell proliferation. These results indicate that the sequence between -298 and +24 consists of the basal promoter activity for TMEPAI. Sp1 promotes TMEPAI expression and contributes to cell proliferation.