The metabolites of vitamin D3 (VD3) mediated by different cytochrome P450 (CYP) enzymes, play fundamental roles in many physiological processes in relation to human health. These metabolites regulate a variety of cellular signal pathways through the direct binding of activated vitamin D receptor/retinoic X receptor (VDR/RXR) heterodimeric complex to specific DNA sequences. Thus, the polymorphisms of VDR and VD3 metabolizing enzymes lead to differentiated efficiency of VD3 and further affect serum VD3 levels. Moreover, VDR activation is demonstrated to inhibit the growth of various cancers, including colorectal cancer. However, excessive intake of vitamin D may lead to hypercalcemia, which limits the application of vitamin D tremendously. In this review, we have summarized the advances in VD3 research, especially the metabolism map of VD3 and the molecular mechanisms of inhibiting growth and inducing differentiation in colorectal cancer mediated by VDR-associated cellular signal pathways. The relationship between VDR polymorphism and the risk of colorectal cancer is also illustrated. In particular, novel pathways of the activation of VD3 started by CYP11A1 and CYP3A4 are highlighted, which produce several noncalcemic and antiproliferative metabolites. At last, the hypothesis is put forward that further research of CYP-mediated VD3 metabolites may develop therapeutic agents for colorectal cancer without resulting in hypercalcemia.