Salmonellosis orSalmonella, one of the most common food-borne diseases, remains a major public health problem worldwide. Intestinal epithelial cells (IECs) play an essential role in the mucosal innate immunity of the host to defend against the invasion ofSalmonellaby interleukin (IL)−8 and human β-defensin-2 (hBD-2). Accumulated research has unravelled important roles of vitamin D in the regulation of innate immunity. Therefore, we investigated the effects of 1,25-dihydroxyvitamin D3 (1,25D3) onSalmonella-induced innate immunity in IECs. We demonstrate that pretreatment of 1,25D3 results in suppression ofSalmonella-induced IL-8 but enhancement of hBD-2, either protein secretion and mRNA expression, in IECs. Furthermore, 1,25D3 enhancedSalmonella-induced membranous recruitment of nucleotide oligomerization domain (NOD2) and its mRNA expression and activation of protein kinase B (Akt), a downstream effector of phosphoinositide 3-kinase (PI3K). Inhibition of the PI3K/Akt signal counteracted the suppressive effect of 1,25D3 onSalmonella-induced IL-8 expression, while knock-down of NOD2 by siRNA diminished the enhanced hBD-2 expression. These data suggest differential regulation of 1,25D3 onSalmonella-induced IL-8 and hBD-2 expression in IECs via PI3K/Akt signal and NOD2 protein expression, respectively. Active vitamin D-enhanced anti-microbial peptide inSalmonella-infected IECs protected the host against infection, while modulation of proinflammatory responses by active vitamin D prevented the host from the detrimental effects of overwhelming inflammation. Thus, active vitamin D-induced innate immunity in IECs enhances the host's protective mechanism, which may provide an alternative therapy for invasiveSalmonellainfection.