Context: There is no effective treatment for systemic sclerosis and related fibrosing diseases. Recently the action of CYP11A1 on vitamin D3 was shown to produce biologically active 20S-hydroxyvitamin D [20(OH)D3] and 20,23(OH)2D3, 20,22(OH)2D3, and 17,20,23(OH)3D3. Objectives: Because 20(OH)D3 is noncalcemic (nontoxic) in vivo at very high doses, we evaluated its antifibrogenic activities both in vitro and in vivo. Because it is further metabolized by CYP11A1, we also tested preclinical utilities of its hydroxyderivatives, especially 20,23(OH)2D3. Design: Human dermal fibroblasts from scleroderma and normal donors were used to test the efficiency of hydroxyvitamin D derivatives in inhibiting TGF-β1-induced collagen and hyaluronan synthesis and inhibiting cell proliferation. The in vivo activity of 20(OH)D3 was tested using bleomycin- induced sclerosis in C57BL/6 mice. Results: 20(OH)D3 and 20,23(OH)2D 3 inhibited TGF-β1-induced collagen and hyaluronan synthesis similarly to 1,25(OH)2D3 in cultured human fibroblasts. Also, 20(OH)D3, 20,23(OH)2D3, and 1,25(OH) 2D3 suppressed TGF-β1-induced expression of COL1A2, COL3A1, and hyaluronan synthase-2 mRNA, indicating that they regulate these matrix components at the transcriptional level. 20(OH)D3, 20,23(OH)2D3, 20,22(OH)2D3, and 17,20,23(OH)3D3 inhibited proliferation of dermal fibroblasts with comparable potency with 1,25(OH)2D3, with 20(OH)D2 being less active and 1α(OH)D3 being almost inactive.20,23(OH) 2D3 at 3μg/kg had no effect on serum Ca++ or fibroblast growth factor-23 levels and did not cause any noticeable signs of morbidity. 20(OH)D3 markedly suppressed fibrogenesis in mice given sc bleomycin as demonstrated by total collagen content and hematoxylin and eosin staining of skin biopsies. Conclusions: 20(OH)D3 is an excellent candidate for preclinical studies on scleroderma, with other CYP11A1-derived products of its metabolism deserving further testing for antibrogenic activity. Copyright © 2013 by The Endocrine Society.