Recurrent hypoglycemia blunts the brain's ability to sense and respond to subsequent hypoglycemic episodes. Glucose-sensing neurons in the ventromedial hypothalamus nucleus (VMN) are well situated to play a role in hypoglycemia detection. VMN glucose-inhibited (GI) neurons, which decrease their firing rate as extracellular glucose increases, are extremely sensitive to decreased extracellular glucose. We hypothesize that recurrent hypoglycemia decreases the glucose sensitivity of VMN GI neurons. To test our hypothesis, 14- to 21-day-old Sprague-Dawley rats were subcutaneously injected with regular human insulin (4 U/kg) or saline (control) for three consecutive days. Blood glucose levels 1 h after insulin injection on day 3 were significantly lower than on day 1, reflecting an impaired ability to counteract hypoglycemia. On day 4, the glucose sensitivity of VMN GI neurons was measured using conventional whole cell current-clamp recording. After recurrent insulin-induced hypoglycemia, VMN GI neurons only responded to a glucose decrease from 2.5 to 0.1, but not 0.5, mM. Additionally, lactate supplementation also decreased glucose sensitivity of VMN GI neurons. Thus our findings suggest that decreases in glucose sensitivity of VMN GI neurons may contribute to the impairments in central glucose-sensing mechanisms after recurrent hypoglycemia.