Sensing and reacting to tissue damage is a fundamental function of immune systems. We previously found that Macrophage inducible C-type lectin (Mincle) is an activating C-type lectin receptor that senses damaged cells. We also found that Mincle also recognizes glycolipid ligands on pathogens, such as mycobacteria. To screen endogenous glycolipids ligands derived from damaged cells, we fractionated supernatants from damaged cells and identified a lipophilic component that activates reporter cells expressing Mincle. Mass spectrometry and NMR spectroscopy identified the component structure as β-glucosylceramide (GlcCer), a ubiquitous intracellular metabolite. Synthetic β-GlcCer activated myeloid cells and induced production of inflammatory cytokines; this production was abrogated in Mincle-deficient cells. Sterile inflammation induced by excessive cell death was exacerbated by hematopoietic-specific deletion of degrading enzyme of β-GlcCer (β-glucosylceramidase, GBA1). However, this enhanced inflammation was ameliorated in a Mincle-deficient background. GBA1–/– dendritic cells (DCs) in which β-GlcCer accumulates triggered antigen-specific T-cell responses more efficiently than WT DCs, whereas this enhancement was eliminated in DCs from GBA1–/– × Mincle–/– mice. These results suggest that β-GlcCer is an endogenous ligand for Mincle and possesses immunostimulatory activity.