Multiple injections of low-dose streptozotocin (MLDS) induce lymphocytic insulitis and diabetes in rodents. To test whether the influx of inflammatory cells was associated with changes in the expression of chemokines, we measured the expression of all known chemokine ligands by real-time quantitative PCR in isolated islets. With the exception of CCL20 and CCL19, chemokines were not significantly expressed in islets from wild-type mice before MLDS treatment. Ten days after treatment, the expression of several chemokines, including CXCL9, CCL1, CXCL10, and CCL21, was dramatically up-regulated. The expression of CCL1, CXCL9, and CCL21 protein was confirmed by immunohistochemistry and was mostly associated with the infiltrating cells. The mouse herpesvirus 68-encoded chemokine decoy receptor M3 can broadly engage these chemokines with high affinity. To test whether a blockade of chemokine function would alter the onset or magnitude of insulitis and diabetes, we used transgenic mice expressing M3 in β cells (rat insulin promoter (RIP)-M3 mice). RIP-M3 mice were normoglycemic and responded normally to glucose challenge but were remarkably resistant to diabetes induced by MLDS. Islets from MLDS-treated RIP-M3 mice had fewer inflammatory cells and expressed lower levels of chemokines than those from MLDS-treated controls. The role of M3 in chemokine blockade during insulitis was further supported by in vitro experiments demonstrating that multiple chemokines up-regulated during islet inflammation are high-affinity M3 ligands that can be simultaneously sequestered. These results implicate chemokines as key mediators of insulitis and suggest that their blockade may represent a novel strategy to prevent insulitis and islet destruction.