Hormonally active vitamin D(3)-1,25-dihydroxyvitamin D(3) (1,25D3)-acts as a signaling molecule in cutaneous immunity by increasing pattern recognition through Toll-like receptor-2 (TLR2), and increasing the expression and function of antimicrobial peptides. Here we show that the actions of 1,25D3 on keratinocyte innate immune responses are influenced by histone acetylation and require the steroid receptor coactivator 3 (SRC3), which mediates inherent histone acetyltransferase (HAT) activity. SRC3 was detected in the suprabasal and granular layer of the skin, similar to cathelicidin expression. HAT activity was important to keratinocyte cathelicidin expression as the combination of histone deacetylase inhibitors (HDACi) (butyrate or trichostatin A) and 1,25D3 increased cathelicidin and CD14 expression and enhanced the antimicrobial function of keratinocytes against Staphylococcus aureus. This treatment, or activation of TLR2, also directly increased acetylation of histone 4. Small interfering RNA silencing of the vitamin D receptor or SRC3 blocked the induction of cathelicidin and CD14 by 1,25D3. HDACi could not reverse this effect or influence cathelicidin in the absence of 1,25D3, suggesting that both are necessary for function. These studies demonstrate that the epigenetic control of gene transcription by histone acetylation is important for 1,25D3-regulated antimicrobial and TLR function of keratinocytes, essential elements of the innate immune response of the skin.