While the neural control of glucoregulatory responses to insulin-induced hypoglycemia is beginning to be elucidated, brain sites responsible for behavioral responses to hypoglycemia are relatively poorly understood. To help elucidate central control mechanisms associated with hypoglycemia unawareness, we first developed an animal model of hypoglycemia-induced behavioral arousal, as a surrogate for "awareness", using a conditioned place preference (CPP) model. We found that hypoglycemia reversed previously acquired CPP in rats and that recurrent hypoglycemia prevented this reversal. Pre-treatment with a brain-penetrant selective orexin receptor-1 antagonist, SB334867A, blocked hypoglycemia-induced reversal of CPP. Recurrently hypoglycemic rats also showed decreased prepro-orexin mRNA expression in the perifornical hypothalamus by 50% but adjacent lateral hypothalamic expression was unaffected. Pre-treatment with sertraline, previously shown to prevent hypoglycemia-associated glucoregulatory failure, did not prevent attenuation of hypoglycemia-induced CPP reversal by recurrent hypoglycemia. This work describes the first behavioral model of hypoglycemia unawareness and suggests a role for orexin neurons in mediating behavioral responses to hypoglycemia.