Interleukin-24 (IL-24) is a novel tumor suppressor/cytokine gene expressed in normal human melanocytes but for which expression is nearly undetectable in metastatic melanoma. Overexpression of the IL-24 protein has been shown to inhibit tumor cell proliferation and induce apoptosis in many melanoma cell lines, and is now considered a tumor suppressor. Erlotinib, a small-molecule epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, has been widely studied for the treatment of human lung cancer and other solid tumors, but the erlotinib-targeted therapy has not been tested in melanoma. The objective of this study is to investigate the potency of erlotinib in suppressing the growth of human melanoma cells and whether IL-24 could enhance the antitumor activity of erlotinib. In cell viability and apoptosis assays, treatment with erlotinib dependently inhibited the growth of different melanoma cell lines and when combined with adenoviral vector-mediated IL-24 gene therapy, a significant increase in cell growth inhibition and apoptosis induction resulted (P<0.05). Immunoblot assay showed that the combination treatment of erlotinib and IL-24 considerably increased the cleavage of caspase-3 and caspase-9 and the expression of Apaf-1 protein in melanoma cells, inducing activation of the Apaf-1-dependent apoptotic pathways. Moreover, this combination treatment markedly inhibited phosphorylation of the EGFR, phosphatidylinositol-3 kinase, and Akt proteins, inactivating the Akt-dependent cell survival signaling pathway. These results show that a combination of IL-24-mediated molecular therapy and EGFR inhibitors such as erlotinib may be a promising treatment strategy for human melanoma and will serve as a basis for guiding the combination treatment designs in future preclinical and clinical trials.