A role for vitamin A in host defense against Mycobacterium tuberculosis has been suggested through epidemiological and in vitro studies; however, the antimicrobial mechanism is unclear. Here, we demonstrate that vitamin A mediates host defense through regulation of cellular cholesterol content. Comparison of monocytes stimulated with all-trans retinoic acid (ATRA) or 1,25-dihydroxyvitamin D3, the biologically active forms of vitamin A and vitamin D respectively, indicates that ATRA and 1,25D3 induce mechanistically distinct antimicrobial activities. Gene expression profiling reveals that ATRA but not 1,25D3 triggers a lipid metabolism and efflux pathway, including expression of lysosomal lipid transport gene NPC2. ATRA-induced decrease in total cellular cholesterol content, subcellular lipid reorganization, lysosomal acidification and antimicrobial activity are all dependent upon expression of NPC2. Finally, the addition of HIV-protease inhibitors known to inhibit cholesterol efflux, Ritonavir and Nelfinavir, blocked both ATRA-induced cholesterol decrease as well as antimicrobial activity. Taken together, these results suggest that the vitamin A-mediated host defense mechanism against M. tuberculosis requires regulation of cellular cholesterol.