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1α,25-Dihydroxyvitamin D₃ (VD₃), the biologically active form of vitamin D, may have either pro- or anti-inflammatory activities because of its diverse actions on nuclear factor kappa B (NF-κB). Previous studies indicated that VD₃ can either activate or inhibit NF-κB via Akt-induced IκBα phosphorylation and increase in IκBα synthesis respectively. At present, the relevant contribution of each mechanism has not been fully explored. We observed a VD₃-mediated NF-κB inhibitory effect in vitamin D receptor (VDR)-positive MCF-7 breast cancer cells. We showed that VD₃ induced VDR-dependent IκBα expression but still able to lead on transient NF-κB p65 nuclear translocation through Akt-induced IκBα phosphorylation. Upon TNFα stimulation, VD₃ was not capable to inhibit IκBα degradation, p65 nuclear translocation and p65/p50-DNA binding. Here, we found that VD₃ strongly repressed p65 transactivation in MCF-7 …