BACKGROUND High fat diet and its induced changes in glucose homeostasis, inflammation and obesity continue to be an epidemic in developed countries. The A2b adenosine receptor (A2bAR) is known to regulate inflammation. We used a diet-induced obesity murine knockout model to investigate the role of this receptor in mediating metabolic homeostasis, and correlated our findings in obese patient samples. RESULTS Administration of high fat, high cholesterol diet (HFD) for sixteen weeks vastly upregulated the expression of the A2bAR in control mice, while A2bAR knockout (KO) mice under this diet developed greater obesity and hallmarks of type 2 diabetes (T2D), assessed by delayed glucose clearance and augmented insulin levels compared to matching control mice. We identified a novel link between the expression of A2bAR, insulin receptor substrate 2 (IRS-2), and insulin signaling, determined by Western blotting for IRS-2 and tissue Akt phosphorylation. The latter is impaired in tissues of A2bAR KO mice, along with a greater inflammatory state. Additional mechanisms involved include A2bAR regulation of SREBP-1 expression, a repressor of IRS-2. Importantly, pharmacological activation of the A2bAR by injection of the A2bAR ligand BAY 60-6583 for four weeks post HFD restores IRS-2 levels, and ameliorates T2D. Finally, in obese human subjects A2bAR expression correlates strongly with IRS-2 expression. CONCLUSIONS Our study identified the A2bAR as a significant regulator of HFD-induced hallmarks of T2D, thereby pointing to its therapeutic potential.