20-Hydroxyvitamin D3 is a derivative of vitamin D3 that exhibits anti proliferative, prodifferentiative and immunomodulatory effects, thus showing promise as a therapeutic drug for the treatment of cancers and inflammatory diseases. To further characterise 20-hydroxyvitamin D3, its metabolism by human liver was investigated and it was found to be metabolised to three products, each with an additional hydroxyl group. The major cytochrome P450 enzyme responsible for these products was CYP3A4, with CYP2R1 playing a lesser role. These metabolic products, present in human serum, displayed stronger inhibition of melanoma cell proliferation than 20-hydroxyvitamin D3. Thus, liver P450s appear to activate 20-hydroxyvitamin D3.