Tumour-initiating cells capable of self-renewal and differentiation, which are responsible for tumour growth, have been identified in human haematological malignancies^, and solid cancers^,,,. If such minority populations are associated with tumour progression in human patients, specific targeting of tumour-initiating cells could be a strategy to eradicate cancers currently resistant to systemic therapy. Here we identify a subpopulation enriched for human malignant-melanoma-initiating cells (MMIC) defined by expression of the chemoresistance mediator ABCB5 (refs , ) and show that specific targeting of this tumorigenic minority population inhibits tumour growth. ABCB5⁺ tumour cells detected in human melanoma patients show a primitive molecular phenotype and correlate with clinical melanoma progression. In serial human-to-mouse xenotransplantation experiments, ABCB5⁺ melanoma cells possess greater tumorigenic capacity than ABCB5^- bulk populations and re-establish clinical tumour heterogeneity. In vivo genetic lineage tracking demonstrates a specific capacity of ABCB5⁺ subpopulations for self-renewal and differentiation, because ABCB5⁺ cancer cells generate both ABCB5⁺ and ABCB5^- progeny, whereas ABCB5^- tumour populations give rise, at lower rates, exclusively to ABCB5^-cells. In an initial proof-of-principle analysis, designed to test the hypothesis that MMIC are also required for growth of established tumours, systemic administration of a monoclonal antibody directed at ABCB5, shown to be capable of inducing antibody-dependent cell-mediated cytotoxicity in ABCB5⁺ MMIC, exerted tumour-inhibitory effects. Identification of tumour-initiating cells with enhanced abundance in more advanced disease but susceptibility to specific targeting through a defining chemoresistance determinant has important implications for cancer therapy.