Recent work suggests that insulin may exert both positive and negative feedback directly on pancreatic β-cells. To investigate the hypothesis that insulin modulates β-cell metabolism, mouse islets and β-cell clusters were loaded with rhodamine 123 to dynamically monitor mitochondrial membrane potential (ΔΨm). Spontaneous oscillations in ΔΨm (period: 218 ± 26 s) were observed in 17 of 30 islets exposed to 11.1 mmol/l glucose. Acute insulin application (100 nmol/l) hyperpolarized ΔΨm, indicating a change in mitochondrial activity. The ATP-sensitive K+ (KATP) channel opener diazoxide or the L-type calcium channel blocker nifedipine mimicked the effect of insulin, suggesting that insulin activates KATP channels to hyperpolarize ΔΨm by inhibiting calcium influx. Treatment with forskolin, which increases endogenous insulin secretion, also mimicked the effect of exogenous insulin, suggesting physiological feedback. Pretreatment with nifedipine or the KATP inhibitor glyburide prevented insulin action, further implicating a KATP channel pathway. Together, these data suggest a feedback mechanism whereby insulin receptor activation opens KATP channels to inhibit further secretion. The resulting reduction in β-cell calcium increases the energy stored in the mitochondrial gradient that drives ATP production. Insulin feedback onto mitochondria may thus help to calibrate the energy needs of the β-cell on a minute-to-minute basis.