Background: Melanoma is highly resistant to current modalities of therapy, with the extent of pigmentation playing an important role in therapeutic resistance. Nuclear factor-B (NF-κB) is constitutively activated in melanoma and can serve as a molecular target for cancer therapy and steroid/secosteroid action. Methods: Cultured melanoma cells were used for mechanistic studies on NF-B activity, utilising immunofluorescence, western blotting, EMSA, ELISA, gene reporter, and estimated DNA synthesis assays. Formalin-fixed, paraffin-embedded specimens from melanoma patients were used for immunocytochemical analysis of NF-B activity in situ.Results:Novel 20-hydroxyvitamin (20(OH)D 3) and classical 1α,25- dihydroxyvitamin D 3 (1,25(OH) 2 D 3) secosteroids inhibited melanoma cell proliferation. Active forms of vitamin D were found to inhibit NF-B activity in nonpigmented cells, while having no effect on pigmented cells. Treatment of nonpigmented cells with vitamin D3 derivatives inhibited NF-B DNA binding and NF-B-dependent reporter assays, as well as inhibited the nuclear translocation of the p65 NF-B subunit and its accumulation in the cytoplasm. Moreover, analysis of biopsies of melanoma patients showed that nonpigmented and slightly pigmented melanomas displayed higher nuclear NF-B p65 expression than highly pigmented melanomas. Conclusion: Classical 1,25(OH) 2D 3 and novel 20(OH)D 3 hydroxyderivatives of vitamin D 3 can target NF-B and regulate melanoma progression in nonpigmented melanoma cells. Melanin pigmentation is associated with the resistance of melanomas to 20(OH)D 3 and 1,25(OH) 2D 3 treatment. © 2011 Cancer Research UK All rights reserved.