Background Interferon-alpha (IFN-alpha) plays a pivotal role in host antitumor immunity, and the evasion of IFN-alpha signaling pathway can lead to IFN-alpha resistance during the treatment of cancer. Although the interplay between IFN-alpha and tumor cells has been extensively investigated in differentiated tumor cells, much less attention has been directed to tumor-repopulating cells (TRCs). Methods Three-dimentional soft fibrin matrix was used to select and grow highly malignant and tumorigenic melanoma TRCs. The regulation of integrin beta 3 (ITGB3)-c-SRC-STAT signaling pathway in melanoma TRCs was investigated both in vitro and in vivo. The relevant mRNA and protein expression levels were analyzed by qRT-PCR and western blot analysis. Immunoprecipitation and chromatin immunoprecipitation (ChIP) followed by qPCR (ChIP-qPCR) assays were performed to detect protein-protein and protein-DNA interactions. The clinical impacts of retinoic acid inducible gene-I (RIG-I) were assessed in melanoma datasets obtained from The Cancer Genome Atlas and Gene Expression Omnibus profiles. Results IFN-alpha -induced apoptosis was decreased in melanoma TRCs. Compared with conventional flask-cultured cells, IFN-alpha -mediated STAT1 activation was diminished in melanoma TRCs. Decreased expression of RIG-I in melanoma TRCs led to diminished activation of STAT1 via enhancing the interaction between Src homology region 2 domain-containing phosphatase-1 and STAT1. In addition, low expression levels of RIG-I correlated with poor prognosis in patients with melanoma. STAT3 was highly phosphorylated in TRCs and knockdown of STAT3 reversed the downregulation of RIG-I in TRCs. Knockdown of STAT3 resulted in STAT1 activation and increased expression of the pro-apoptosis genes in IFN-alpha -treated TRCs. Combined treatment of STAT3 inhibitor and IFN-alpha increased the apoptosis rate of TRCs. Disruption of ITGB3/c-SRC/STAT3 signaling pathway significantly elevated the efficiency of IFN-alpha -induced apoptosis of TRCs. Conclusions In melanoma TRCs, ITGB3-c-SRC-STAT3 pathway caused RIG-I repression and then affect STAT1 activation to cause resistance to IFN-alpha -induced apoptosis. RIG-I is a prognostic marker in patients with melanoma. Combination of STAT3 inhibitor and IFN-alpha could enhance the efficacy of melanoma treatment. Our findings may provide a new concept of combinatorial treatment for future immunotherapy.