Interleukin-24 (IL-24) has been shown to be a tumor-suppressor gene and the protein product found to be constitutively expressed by melanocytes, nerve cells, and some primary melanomas. The potential effect of adenovirus (AdV)-mediated IL-24 gene therapy was explored on human pancreatic carcinoma by using a pancreatic carcinoma cell line, patu8988. A recombinant adenovirus, AdVGFP/IL-24, expressing the marker, green fluorescent protein (GFP), and the tumor-suppressor gene, IL-24, was constructed. AdVGFP/IL-24 treatment of pancreatic carcinoma cells in vitro significantly induced pancreatic carcinoma cell cytotoxicity and apoptosis, compared with AdVGFP without IL-24 expression. In nude mice bearing patu8988 tumors, intratumoral injections of AdVGFP/IL-24 significantly inhibited pancreatic carcinoma growth. In addition, the molecular mechanism of tumor suppression was elucidated by downregulating the expression of vascular endothelial growth factor, CD34, and Bcl-2, as well as inhibiting tumor angiogenesis. Therefore, AdVGFP/IL-24 has the potential to serve as a novel tool for pancreatic carcinoma gene therapy.