Scope Vitamin D(3)is a critical molecule for the properly controlled activity of the immune system. In myeloid-derived cells, vitamin D(3)induces the production of the antimicrobial and antitumor peptide cathelicidin. In this study, the mechanism of the entry of 25-hydroxycholecalciferol (25(OH)D) in myeloid-derived cells is explored. Methods and results Here, a novel regulatory pathway of vitamin D(3)biology is described. Using a polyclonal antibody, two different chemical inhibitors, and a high-density lipoprotein as a competing ligand, it is demonstrated here that the 25(OH)D signaling pathway in myeloid cells depends on scavenger receptor class B type I (SR-B1). This effect is observed in the THP-1 monocytic cell line and in human primary monocytes. SR-B1 blockade abrogates the cellular uptake of 25(OH)D leading to a general shut down of the gene transcription program modulated by 25(OH)D. The results obtained at the transcriptional level are confirmed at the protein and functional level for CD14 in the THP-1 cell line. Conclusion In conclusion, SR-B1 plays a critical role in vitamin D(3)biology, paving the way for novel therapeutic interventions.