<p>Despite significant advancements in diagnosis and treatment, cancer remains a leading cause of mortality globally. Cancer gene therapy has emerged as a promising strategy, with numerous clinical trials demonstrating its efficacy in targeting tumor cells, vasculature, and immune components. However, precise and selective gene expression regulation remains a challenge. In this study, we demonstrated that the CREPT (cell-cycle related and expression-elevated protein in tumor) promoter holds great potential for targeted cancer gene therapy. CREPT, a tumor-promoting protein as a positive regulator of gene transcription, is highly expressed across various cancer types while exhibiting minimal expression in normal tissues. The CREPT promoter mediated robust and tumor-selective transgene expression in vivo following both local and systemic administration, with only minimal off-target expression detected in blood and none in normal organs. Leveraging this specificity, we engineered an adenovirus encoding diphtheria toxin fragment A under CREPT promoter regulation. This construct was selectively expressed and inhibited protein synthesis, leading cancer cell death in vitro and in vivo. These findings demonstrate that the CREPT promoter may serve as a useful regulatory element for the development of targeted cancer gene therapy.</p>