Background: Hepatocellular carcinoma (HCC) has a dismal 5-year-survival rate of 10%, so novel strategies are warranted. IL-24 mediates anti-tumor activity reducing STAT3 expression, which suggests that interferon (IFN) alpha may augment tumor cell lysis and reduce angiogenesis. We investigated the antitumor activity of treatment with IFN-α, with the oncolytic adenovirus SG600-IL-24, or the combination of both in HCC in vitro and in vivo.Results: RT-PCR, ELISA assay and Western-blot confirmed that the exogenous IL-24 gene was highly expressed in HCC cells infected with SG600-IL-24. Treatment with combined IFN-α and SG600-IL-24 suppressed growth and promoted apoptosis of the HepG2, MHCC97L, and HCCLM3 cell lines compared with the normal cell line L02. The combined therapy increased STAT1 and SOCS1 and apoptosis, but decreased the expression of the metastatic and angiogenic proteins MMP-2, XIAP, OPN, and VEGF, which are regulated by STAT3 in HCC cells in vitro. To assess the effects in vivo, the HCC cell line HCCLM3 was transplanted subcutaneously into the right flanks of nude mice. Mice in the IFN-α group, the SG600-IL-24 group, or the combined therapy group had significantly suppressed growth of the HCC xenografted tumors compared to the PBS control group of mice. Among the mice treated with the combination of IFN-α and SG600-IL-24, three of those eight mice had long-term survival and no evidence of a tumor. These mice also had decreased expression of the metastatic and angiogenic proteins MMP-2, XIAP, OPN, and VEGF.Conclusions: The present study demonstrated for the first time the potential antitumor activity of IFN-α combined with the oncolytic adenovirus SG600-IL-24 in HCC both in vitro and in vivo, and suggests its further development as a potential candidate for HCC cancer gene therapy. © 2012 Wang et al.; licensee BioMed Central Ltd.