BACKGROUND. Besides their health benefits, dietary omega-3 fatty acids (n-3 PUFAs) can impair host resistance to intracellular pathogens. Previously, we and others have showed that n-3 PUFA-treated macrophages poorly control Mycobacterium tuberculosis infection in vitro. METHODS. Wild-type and fat-1 transgenic mice were infected with virulent H37Rv M. tuberculosis via the aerosol route. We evaluated bacteriological and histopathological changes in lungs, as well as differences in activation and antimycobacterial capacity in primary macrophages ex vivo. RESULTS. fat-1 mice were more susceptible to tuberculosis, as demonstrated by higher bacterial loads and less robust inflammatory responses in lungs. Macrophages obtained from fat-1 mice were more readily infected with M. tuberculosis in vitro, compared with wild-type macrophages. This impaired bacterial control in cells from fat-1 mice correlated with reduced proinflammatory cytokine secretion, impaired oxidative metabolism, and diminished M. tuberculosis-lysotracker colocalization within phagosomes. CONCLUSIONS. We showed that endogenous production of n-3 PUFAs in fat-1 mice increases their susceptibility to tuberculosis, which could be explained in part by diminished activation and antimycobacterial responses in cells from fat-1 mice. These data suggest that n-3 PUFA-supplemented diets might have a detrimental effect on immunity to M. tuberculosis and raise concerns regarding the safety of omega-3 dietary supplementation in humans.