Integrins are components of cell-matrix adhesions, and function as scaffolds for various signal transduction pathways. So far no lipid ligand for integrin has been reported. Here we show that a lipid, oxysterol 25-hydroxycholesterol (25HC), directly binds to α5β1 and αvβ3 integrins to activate integrin-focal adhesion kinase (FAK) signaling. Treatment of macrophages and epithelial cells with 25HC results in an increase in activated αvβ3 integrin in podosome and focal adhesion matrix adhesion sites. Moreover, activation of pattern recognition receptor on macrophages induces secretion of 25HC, triggering integrin signaling and the production of proinflammatory cytokines such as TNF and IL-6. Thus, the lipid molecule 25HC is a physiologically relevant activator of integrins and is involved in positively regulating proinflammatory responses. Our data suggest that extracellular 25HC links innate immune inflammatory response with integrin signaling.