The active vitamin D metabolite 1,25-dihydroxyvitamin D3 (1,25(OH)2 D3 ) inhibits proliferation and promotes differentiation of colon cancer cells through the activation of vitamin D receptor (VDR), a transcription factor of the nuclear receptor superfamily. Additionally, 1,25(OH)2 D3 has several nongenomic effects of uncertain relevance. We show that 1,25(OH)2 D3 induces a transcription-independent Ca2+ infl ux and activation of RhoA- Rho-associated coiled kinase (ROCK). This requires VDR and is followed by activation of the p38 mitogen-activated protein kinase (p38MAPK) and mitogen- and stress- activated kinase 1 (MSK1). As shown by the use of chemical inhibitors, dominant-negative mutants and small interfering RNA, RhoA - ROCK, and p38MAPK-MSK1 activation is necessary for the induction of CDH1 /E-cadherin, CYP24 , and other genes and of an adhesive phenotype by 1,25(OH)2 D3 . RhoA - ROCK and MSK1 are also required for the inhibition of Wnt -β-catenin pathway and cell proliferation. Thus, the action of 1,25(OH) 2 D 3 on colon carcinoma cells depends on the dual action of VDR as a transcription factor and a nongenomic activator of RhoA - ROCK and p38MAPK-MSK1. © 2008 Ordóñez-Morán et al.