Human gingival epithelial cells (GEC) produce peptides, such as β-defensins and the cathelicidin LL-37, that are both antimicrobial and that modulate the innate immune response. In myeloid and airway epithelial cells, the active form of vitamin D(3) [1,25(OH)(2)D(3)] increases the expression and antibacterial activity of LL-37. To examine the activity of vitamin D on the innate immune defense of the gingival epithelium, cultured epithelial cells were treated with either 10(-8) M 1,25(OH)(2)D(3) or ethanol for up to 24 h. A time-dependent induction of LL-37 mRNA up to 13-fold at 24 h in both standard monolayer and three-dimensional cultures was observed. Induction of the vitamin D receptor and the 1-α-hydroxylase genes was also observed. The hydroxylase was functional, as LL-37 induction was observed in response to stimulation by 25(OH)D(3). Through microarray analysis of other innate immune genes, CD14 expression increased 4-fold, and triggering receptor expressed on myeloid cells-1 (TREM-1) was upregulated 16-fold after 24 h of treatment with 1,25(OH)(2)D(3). TREM-1 is a pivotal amplifier of the innate immune response in macrophages, leading to increased production by inflammatory response genes. Activation of TREM-1 on the GEC led to an increase in interleukin-8 (IL-8) mRNA levels. Incubation of three-dimensional cultures with 1,25(OH)(2)D(3) led to an increase in antibacterial activity against the periodontal pathogen Aggregatibacter actinomycetemcomitans when the bacteria were added to the apical surface. This study is the first to demonstrate the effect of vitamin D on antibacterial defense of oral epithelial cells, suggesting that vitamin D(3) could be utilized to enhance the innate immune defense in the oral cavity.