Osteopontin (OPN) is a secreted acidic phosphoprotein that is involved in many inflammatory and immune-modulating disorders. We previously demonstrated that OPN is a novel islet protein and a pro survival factor that may serve as an intrinsic feedback regulator of nitric oxide signaling in β-cells. Here, we investigated the endogenous expression of pancreatic OPN in non obese diabetic (NOD) mice and explored its regulation in the islets and b-cells. High levels of pancreatic OPN mRNA and protein were seen in the prediabetic NOD mice pancreata. The temporal pattern of OPN expression inversely correlated with progression of insulitis and β-cell destruction. Immunostaining of pancreatic serial sections showed co localization of OPN with most of the islet hormones. Next we investigated the regulation of OPN in the islets and β-cells. Naturally occurring early upregulation of OPN transcription was seen after exposure of native normoglycemic NOD islets and β-cells to a high-dose combination of IL-1β, TNF-α and IFN-γ. To distinguish between the effect of cytokines and high glucose on OPN transcription, RINm5F cells were transfected with luciferase-labeled rat OPN promoter and treated with cytokines or glucose. Cytokines induced upregulation of OPN promoter activity within one hour, while glucose induced a dose-dependent upregulation of OPN promoter activity after 24 hrs. Long-term exposures to cytokines or glucose reduced OPN expression and promoter activity. Our data provide the first observations into the presence of a positive intrinsic mechanism that regulates pancreatic OPN expression. Based upon previous studies that support a protective role of OPN in the islets, our data suggest that exhaustion of this local OPN system is implicated in the associated loss of endogenous islet protection and progression of the destructive insulitis and diabetes severity in the NOD mouse model.