Acetyl CoA carboxylase 1 (ACC1) is being investigated as a target for treatment of obesity-associated dyslipidemia and insulin resistance. To investigate the effects of ACC1 inhibition on insulin secretion, three small interfering RNA (siRNA) duplexes targeting ACC1 (siACC1) were transfected into the INS-1-derived cell line 832/13; the most efficacious duplex was also cloned into an adenovirus and used to transduce isolated rat islets. Delivery of the siACC1 duplexes decreased ACC1 mRNA by 60-80% in 832/13 cells and islets, and enzyme activity by 46% compared with cells treated with a non-targeted siRNA. Delivery of siACC1 decreased glucose-stimulated insulin secretion (GSIS) by 70% in 832/13 cells and 33% in islets. Surprisingly, siACC1 treatment decreased glucose oxidation by 49%, and ATP:ADP ratio by 52%, accompanied by clear decreases in pyruvate cycling activity and TCA cycle intermediates. Exposure of siACC1-treated cells to the pyruvate cycling substrate dimethylmalate restored GSIS to normal without recovery of the depressed ATP:ADP ratio. In siACC1-treated cells, glucokinase (GK) protein levels were decreased by 25%, correlated with a 36% decrease in glycogen synthesis and a 33% decrease in glycolytic flux. Furthermore, acute addition of the ACC1 inhibitor TOFA to ß-cells suppressed [14C] glucose incorporation into lipids but had no effect on GSIS, whereas chronic TOFA administration suppressed GSIS and glucose metabolism. In sum, chronic, but not acute suppression of ACC1 activity impairs GSIS via inhibition of glucose rather than lipid metabolism. These findings raise concerns about the use of ACC inhibitors for diabetes therapy.