Invariant natural killer T (iNKT) cells are a unique subset of T lymphocytes that have been implicated in diverse immune reactions, ranging from self-tolerance and development of autoimmunity to responses to pathogens and tumors. Although some degree of autoreactivity of iNKT cells has been shown, it remained controversial whether the T-cell antigen receptor expressed by these cells could recognize microbial antigens, hampering the investigation of their physiological role during tolerance and immunity. Several recent publications have now defined natural antigens for the majority of iNKT cells in some Proteobacteria and in Borrelia burgdorferi, demonstrating specificity of these cells for microbes in addition to self-reactivity. The characterization of natural antigens from bacteria, and the iNKT cell response to bacteria containing them, are decisive steps toward the clarification of the natural role of iNKT cells in host defense against pathogens, and will likely spur numerous findings in the near future.