The short heterodimer partner (SHP) (NR0B2) is an orphan nuclear receptor whose function in pancreatic beta-cells is unclear. Mitochondrial uncoupling protein (UCP2) in beta-cells is upregulated in obesity-related diabetes, causing impaired glucose-stimulated insulin secretion (GSIS). We investigated whether SHP plays a role in UCP2-induced GSIS impairment. We overexpressed SHP in normal islet cells and in islet cells overexpressing UCP2 by an adenovirus-mediated infection technique. We found that SHP overexpression enhanced GSIS in normal islets, and restored GSIS in UCP2-overexpressing islets. SHP overexpression increased the glucose sensitivity of ATP-sensitive K+ (KATP) channels and enhanced the ATP/ADP ratio. A peroxisome proliferator-activated receptor gamma (PPARgamma) antagonist, GW9662, did not block the SHP effect on GSIS. SHP overexpression also corrected the impaired sensitivity of UCP2-overexpressing beta-cells to methylpyruvate, another energy fuel that bypasses glycolysis and directly enters the Krebs cycle. KATP channel inhibition mediated by dihydroxyacetone, which gives reducing equivalents directly to complex II of the electron transport system, was similar in Ad-Null-, Ad-UCP2- and Ad-UCP2+Ad-SHP-infected cells. The mitochondrial metabolic inhibitor sodium azide totally blocked the effect of SHP overexpression on GSIS. These results suggest that SHP positively regulates GSIS in beta-cells and restores glucose sensitivity in UCP2-overexpressing beta-cells by enhancing mitochondrial glucose metabolism, independent of PPARgamma activation.