Vitamin D deficiency is associated with susceptibility to tuberculosis, and its biologically active metabolite, 1α, 25 dihydroxyvitamin D 3 (1α, 25 (OH) 2 D 3), has pleiotropic immune effects. The mechanisms by which 1α, 25 (OH) 2 D 3 protects against tuberculosis are incompletely understood. 1α, 25 (OH) 2 D 3 reduced the growth of mycobacteria in infected human PBMC cultures in a dose-dependent fashion. Coculture with agonists or antagonists of the membrane or nuclear vitamin D receptors indicated that these effects were primarily mediated by the nuclear vitamin D receptors. 1α, 25 (OH) 2 D 3 reduced transcription and secretion of protective IFN-γ, IL-12p40, and TNF in infected PBMC and macrophages, indicating that 1α, 25 (OH) 2 D 3 does not mediate protection via these cytokines. Although NOS2A was up-regulated by 1α, 25 (OH) 2 D 3, inhibition of NO formation marginally affected the suppressive effect of 1α, 25 (OH) 2 D 3 on bacillus Calmette Guérin in infected cells. By contrast, 1α, 25 (OH) 2 D 3 strongly up-regulated the cathelicidin hCAP-18 gene, and some hCAP-18 polypeptide colocalized with CD14 in 1α, 25 (OH) 2 D 3 stimulated PBMC, although no detectable LL-37 peptide was found in supernatants from similar 1α, 25 (OH) 2 D 3-stimulated PBMC cultures. A total of 200 μg/ml of the active peptide LL-37, in turn, reduced the growth of Mycobacterium tuberculosis in culture by 75.7%. These findings suggest that vitamin D contributes to protection against TB by “nonclassical” mechanisms that include the induction of antimicrobial peptides.