In pancreatic ss-cells, metabolic coupling factors generated during glucose metabolism and pyruvate cycling, through anaplerosis/cataplerosis processes, contribute to the regulation of insulin secretion. Pyruvate/citrate cycling across the mitochondrial membrane leads to the production of malonyl-CoA and NADPH, two candidate coupling factors. In order to examine the implication of pyruvate/citrate cycling in glucose-induced insulin secretion (GIIS), different steps of the cycle were inhibited in INS 832/13 cells by pharmacological inhibitors and/or RNAi technology: mitochondrial citrate export, ATP-citrate lyase (ACL) and cytosolic malic enzyme (ME1). The inhibitors of the di- and tri-carboxylate carriers, n-butylmalonate and 1,2,3-benzenetricarboxylate, respectively, reduced GIIS indicating the importance of trans-mitochondrial transport of tri- and dicarboxylates in the action of glucose. To directly test the role of ACL and ME1 in GIIS, shRNA were used to selectively decrease ACL or ME1 expression in transfected INS 832/13 cells. shRNA-ACL reduced ACL protein levels by 67% and this was accompanied by a reduction in GIIS. The amplification/K(ATP)-independent pathway of GIIS was affected by RNAi knockdown of ACL. The ACL inhibitor radicicol also curtailed GIIS. shRNA-ME1 reduced ME1 activity by 62% and decreased GIIS. RNAi-suppression of either ACL or ME1 did not affect glucose oxidation. However, since ACL is required for malonyl-CoA formation, inhibition of ACL expression by shRNA-ACL decreased glucose incorporation into palmitate and increased fatty acid oxidation in INS 832/13 cells. Taken together, the results underscore the importance of pyruvate/citrate cycling in pancreatic ss-cell metabolic signaling and the regulation of GIIS.