Given the anti-inflammatory effects of insulin in human and animal studies done in vivo and given the signaling pathways in common between insulin and the protease-activated receptor 2 (PAR(2)), a G protein-coupled receptor, we hypothesized that insulin would have an impact on the inflammatory actions of PAR(2). We found that low doses or concentrations of insulin in the subnanomolar range reduced PAR(2)-induced inflammation in a murine paw edema model, attenuated PAR(2)-induced leukocyte trafficking in mouse intestinal venules, and reduced PAR(2) calcium signaling in cultured dorsal root ganglion neurons and endothelial cells. This effect of insulin to attenuate PAR(2)-mediated inflammation was reversed when cells were preincubated with LY294002 (a PI3K inhibitor) and GF 109203X (a pan-protein kinase C inhibitor). The enhanced inflammatory effect of PAR(2) observed in vivo in an insulin-deficient murine type 1 diabetes model was attenuated by the local administration of insulin at the inflammatory site. Our data point to an anti-inflammatory action of insulin that targets the acute innate inflammatory response triggered by PAR(2).