The mycobacterial cord factor trehalose-6,6-dimycolate (TDM) and its synthetic analog trehalose-6,6-dibehenate (TDB) are potent adjuvants for Th1/Th17 vaccination that activate Syk-Card9 signaling in APCs. In this study, we have further investigated the molecular mechanism of innate immune activation by TDM and TDB. The Syk-coupling adapter protein FcRgamma was essential for macrophage activation and Th17 adjuvanticity. The FcRgamma-associated C-type lectin receptor Mincle was expressed in macrophages and upregulated by TDM and TDB. Recombinant Mincle-Fc fusion protein specifically bound to the glycolipids. Genetic ablation of Mincle abolished TDM/TDB-induced macrophage activation and induction of T cell immune responses to a tuberculosis subunit vaccine. Macrophages lacking Mincle or FcRgamma were impaired in the inflammatory response to Mycobacterium bovis bacillus Calmette-Guérin. These results establish that Mincle is a key receptor for the mycobacterial cord factor and controls the Th1/Th17 adjuvanticity of TDM and TDB.