Differential expression of the desired gene product in the target tissue is central to the concept of gene therapy. One approach is to use a tissue-specific promoter to drive therapeutic genes, such as the p53 tumor suppressor gene. To determine the feasibility of tissue-specific gene therapy for prostate cancer using prostate specific antigen (PSA) promoter and/or enhancer, in this study, we developed a tissue specific expression vector using a PSA promoter and enhancer. Our results showed that the cloned PSA promoter actively drives gene expression in the PSA-producing prostate cancer cell line (LNCaP). However, barely any promoter activity was detected in the non-PSA producing prostate cancer cell lines (DU145, PC-3) or the non-prostate cell lines (HEK-293, SAOS-2). The wild-type p53 gene driven by this PSA-promoter efficiently suppressed the growth of LNCaP. Moreover, p53 driven by the PSA enhancer-promoter cassette more efficiently suppressed the growth of the PSA-producing prostate cancer cell line (LNCaP) in vitro. This suggest that we were able to manage the tissue specificity by PSA enhancer and promoter. Additionally, the juxtaposed enhancer-promoter cassette showed great enhancement of p53 expression and apoptosis in vitro. Taken together, these results show that PSA enhancer-promoter may be a potential tool for gene therapy for prostate cancer.