1 alpha, 20S-Dihydroxyvitamin D3 [1,20S(OH)(2)D-3], a natural and bioactive vitamin D3 metabolite, was chemically synthesized for the first time. X-ray crystallography analysis of intermediate 15 confirmed its 1 alpha-OH configuration. 1,20S(OH)(2)D-3 interacts with the vitamin D receptor (VDR), with similar potency to its native ligand, 1 alpha, 25-dihydroxyvitamin D-3 [1,25(OH) 2D3] as illustrated by its ability to stimulate translocation of the VDR to the nucleus, stimulate VDRE-reporter activity, regulate VDR downstream genes (VDR, CYP24A1, TRPV6 and CYP27B1), and inhibit the production of inflammatory markers (IFN gamma and IL1 beta). However, their co-crystal structures revealed differential molecular interactions of the 20S-OH moiety and the 25-OH moiety to the VDR, which may explain some differences in their biological activities. Furthermore, this study provides a synthetic route for the synthesis of 1,20S(OH)(2)D-3 using the intermediate 1 alpha, 3 beta-diacetoxypregn-5-en-20-one (3), and provides a molecular and biological basis for the development of 1,20S(OH)(2)D-3 and its analogs as potential therapeutic agents.