To test the hypothesis that glucokinase is a critical regulator of neuronal glucosensing, glucokinase activity was increased, using a glucokinase activator drug, or decreased, using RNA interference combined with calcium imaging in freshly dissociated ventromedial hypothalamic nucleus (VMN) neurons or primary ventromedial hypothalamus (VMH; VMN plus arcuate nucleus) cultures. To assess the validity of our approach, we first showed that glucose-induced (0.5–2.5 mmol/l) changes in intracellular Ca²⁺ concentration ([Ca²⁺]_i) oscillations, using fura-2 and changes in membrane potential (using a membrane potential–sensitive dye), were highly correlated in both glucose-excited and -inhibited neurons. Also, glucose-excited neurons increased (half-maximal effective concentration [EC₅₀] = 0.54 mmol/l) and glucose-inhibited neurons decreased (half-maximal inhibitory concentration [IC₅₀] = 1.12 mmol/l) [Ca²⁺]_i oscillations to incremental changes in glucose from 0.3 to 5 mmol/l. In untreated primary VMH neuronal cultures, the expression of glucokinase mRNA and the number of demonstrable glucosensing neurons fell spontaneously by half over 12–96 h without loss of viable neurons. Transfection of neurons with small interfering glucokinase RNA did not affect survival but did reduce glucokinase mRNA by 90% in association with loss of all demonstrable glucose-excited neurons and a 99% reduction in glucose-inhibited neurons. A pharmacological glucokinase activator produced a dose-related increase in [Ca²⁺]_i oscillations in glucose-excited neurons (EC₅₀ = 0.98 mmol/l) and a decrease in glucose-inhibited neurons (IC₅₀ = 0.025 μmol/l) held at 0.5 mmol/l glucose. Together, these data support a critical role for glucokinase in neuronal glucosensing.