Active forms of vitamin D3, including , 20(OH)D3 and , inhibited cell proliferation, migration rate and the ability to form colonies and spheroids in the wild-type melanoma cell line, while cells with the vitamin D receptor (VDR) silenced showed an increased but not complete resistance to their action. Furthermore, silencing of the VDR in melanoma cells enhanced their proliferation as well as spheroid and colony formation and increased their migration rate. Previous clinicopathological studies have shown an inverse correlation between VDR expression, melanoma progression and poor outcome of the disease. Thus, the expression of VDR is not only necessary for the inhibition of melanoma growth by active forms of vitamin D, but the VDR can also function as a melanoma tumor suppressor gene. Vitamin D3 is not only involved in calcium and phosphate metabolism in humans, but it can also affect proliferation and differentiation of normal and cancer cells, including melanoma. The mechanism of the anti-cancer action of vitamin D3 is not fully understood. The nuclear vitamin D receptor (VDR) is crucial for the phenotypic effects of vitamin D hydroxyderivatives. VDR expression shows an inverse correlation with melanoma progression and poor outcome of the disease. In this study we knocked out the VDR in a human melanoma cell line using CRISPR methodology. This enhanced the proliferation of melanoma cells grown in monolayer culture, spheroids or colonies and their migration. Activated forms of vitamin D, including classical , 20(OH)D3 and , inhibited cell proliferation, migration rate and the ability to form colonies and spheroids in the wild-type melanoma cell line, while VDR KO cells showed a degree of resistance to their action. These results indicate that expression of VDR is important for the inhibition of melanoma growth induced by activated forms of vitamin D. In conclusion, based on our previous clinicopathological analyses and the current study, we suggest that the VDR can function as a melanoma tumor suppressor gene.