The active form of vitamin D, 1,25-dihydroxyvitamin D (1,25(OH)(2)D) enhances innate immunity by inducing the cathelicidin antimicrobial peptide (hCAP). In monocytes/macrophages, this occurs primarily in response to activation of TLR, that induce expression of the vitamin D receptor and localized synthesis of 1,25(OH)(2)D from precursor 25-hydroxyvitamin D(3) (25OHD). To clarify the relationship between vitamin D and innate immunity, we assessed changes in hCAP expression in vivo and ex vivo in human subjects attending a bone clinic (n = 50). Of these, 38% were vitamin D-insufficient (<75 nM 25OHD) and received supplementation with vitamin D (50,000 IU vitamin D(2) twice weekly for 5 wk). Baseline 25OHD status or vitamin D supplementation had no effect on circulating levels of hCAP. Therefore, ex vivo changes in hCAP for each subject were assessed using peripheral blood monocytes cultured with 10% autologous serum (n = 28). Under these vitamin D "insufficient" conditions the TLR2/1 ligand 19 kDa lipopeptide or the TLR4 ligand LPS, monocytes showed increased expression of the vitamin D-activating enzyme CYP27b1 (5- and 5.5-fold, respectively, both p < 0.01) but decreased expression of hCAP mRNA (10-fold and 30-fold, both p < 0.001). Following treatment with 19 kDa, expression of hCAP: 1) correlated with 25OHD levels in serum culture supplements (R = 0.649, p < 0.001); 2) was significantly enhanced by exogenous 25OHD (5 nM); and 3) was significantly enhanced with serum from vivo vitamin D-supplemented patients. These data suggest that a key role of vitamin D in innate immunity is to maintain localized production of antibacterial hCAP following TLR activation of monocytes.