We have examined effects of the 20,23-dihydroxyvitamin D3 (20,23(OH)2D3), on differentiation and proliferation of human keratinocytes and the anti-inflammatory potential of 20,23(OH)2D3 from its action on nuclear factor-κB (NF-κB). 20,23(OH)2D3 inhibited growth of keratinocytes with a potency comparable to that for 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Cell cycle analysis showed that this inhibition was associated with G1/G0 and G2/M arrests. 20,23(OH)2D3 stimulated production of involucrin mRNA and inhibited production of cytokeratin 14 mRNA in a manner similar to that seen for 1,25(OH)2D3. Flow cytometry showed that these effects were accompanied by increased involucrin protein expression, and an increase in the cell size and granularity. Silencing of the vitamin D receptor (VDR) by corresponding siRNA abolished the stimulatory effect on involucrin gene expression demonstrating an involvement of VDRin 20,23(OH)2D3 action. This mode of action was further substantiated by stimulation of CYP24 gene expression and stimulation of the CYP24 promoter-driven reporter gene activity. 20,23(OH)2D3 displayed several fold lower potency for induction of CYP24 gene expression than 1,25(OH)2D3. Finally, 20,23(OH)2D3 inhibited the transcriptional activity of NF-κB in keratinocytes as demonstrated by EMSA, NF-κB-driven reporter gene activity assays and measurements of translocation of p65 from the cytoplasm to the nucleus. These inhibitory effects were connected with stimulation of the expression of IκBα with subsequent sequestration of NF-κB in the cytoplasm and consequent attenuation of transcriptional activity. In summary, we have characterized 20,23(OH)2D3 as a novel secosteroidal regulator of keratinocytes proliferation and differentiation and a modifier of their immune activity. © 2009 Wiley-Liss, Inc.