We have previously reported that glucose-stimulated insulin secretion (GSIS) is tightly correlated with pyruvate carboxylase (PC)-catalyzed anaplerotic flux into the TCA cycle and stimulation of pyruvate cycling activity. To further evaluate the role of PC in ss-cell function, we constructed a recombinant adenovirus containing an siRNA specific to PC (Ad-siPC). Ad-siPC reduced PC mRNA levels by 83% and 64% and PC protein by 56% and 35% in INS-1-derived 832/13 cells and primary rat islets, respectively. Surprisingly, this manipulation did not impair GSIS in rat islets. In Ad-siPC-treated 832/13 cells, GSIS was slightly increased, while glycolytic rate and glucose oxidation were unaffected. Flux through PC at high glucose was decreased by only 20%, suggesting an increase in PC specific activity. Acetyl carnitine, a surrogate for acetyl CoA, an allosteric activator of PC, was increased by 36% in Ad-siPC-treated cells, suggesting a mechanism by which PC enzymatic activity is maintained with suppressed PC protein levels. In addition, the NADPH:NADP ratio, a proposed coupling factor for GSIS, was unaffected in Ad-siPC-treated cells. We conclude that ss-cells activate compensatory mechanisms in response to suppression of PC expression that prevent impairment of anaplerosis, pyruvate cycling, NAPDH production, and GSIS.