Previous studies have shown that interleukin (IL)-24 as a novel tumor suppressor gene has tumor-suppressor activity in a broad spectrum of human cancer cells both in vitro and in vivo. In this study, we explored the potential effect of adenovirus-mediated IL-24 gene therapy on human hepatocellular carcinoma (HCC) by using a HCC cell line, SMMC-7721. We constructed a recombinant adenovirus, AdVGFP/IL-24 expressing the marker green fluorescent protein (GFP) and the tumor-suppressor gene, IL-24. We demonstrated that AdVGFP/IL-24 treatment of SMMC-7721 cells in vitro significantly induced HCC cell cytotoxicity and apoptosis, and altered HCC cell cycling with an S-phase reduction and G2/M phase arrest, compared with AdVGFP, without IL-24 expresssion (p < 0.05). Furthermore, we also showed that the treatment of SMMC-7721 tumors by an intratumoral injection of AdVGFP/IL-24 significantly suppressed in vivo HCC growth in athymic nude mice, compared with AdVGFP treatment (p < 0.05). In addition, we also elucidated the molecular mechanism responsible for AdVGFP/IL-24-associated tumor suppression. These include: (1) upregulation of p53-independent apoptosis-associated caspase-3 and (2) downregulation of angiogenesis-associated vascular endothelial growth factor and CD34. Therefore, this study will provide a framework for future clinical applications of AdVGFP/IL-24 in HCC gene therapy.