Invariant natural killer T (iNKT) cells are an important source of both T helper type 1 (Th1) and Th2 cytokines, through which they can exert beneficial, as well as deleterious, effects in a variety of inflammatory diseases. This functional heterogeneity raises the question of how far phenotypically distinct subpopulations are responsible for such contrasting activities. In this study, we identify a particular set of iNKT cells that lack the NK1.1 marker (NK1.1(neg)) and secrete high amounts of interleukin (IL)-17 and low levels of interferon (IFN)-gamma and IL-4. NK1.1(neg) iNKT cells produce IL-17 upon synthetic (alpha-galactosylceramide [alpha-GalCer] or PBS-57), as well as natural (lipopolysaccharides or glycolipids derived from Sphingomonas wittichii and Borrelia burgdorferi), ligand stimulation. NK1.1(neg) iNKT cells are more frequent in the lung, which is consistent with a role in the natural immunity to inhaled antigens. Indeed, airway neutrophilia induced by alpha-GalCer or lipopolysaccharide instillation was significantly reduced in iNKT-cell-deficient Jalpha18(-/-) mice, which produced significantly less IL-17 in their bronchoalveolar lavage fluid than wild-type controls. Furthermore, airway neutrophilia was abolished by a single treatment with neutralizing monoclonal antibody against IL-17 before alpha-GalCer administration. Collectively, our findings reveal that NK1.1(neg) iNKT lymphocytes represent a new population of IL-17-producing cells that can contribute to neutrophil recruitment through preferential IL-17 secretion.