Repeated exposure to hypoglycemia leads to suppression of normal counter-regulatory responses (CRR) to hypoglycemia; increasing severe hypoglycemia risk. The mechanism underlying this is unknown, but believed to result primarily from defective glucose-sensing within the ventromedial hypothalamus (VMH). Hypoglycemia evokes a significant systemic and local stress response with an increase in inflammatory mediators such as interleukin-6 (IL-6). In this study, we sought to determine if IL-6 affected hypothalamic glucose-sensing by examining its action on hypoglycemia-induced changes in membrane potential and AMPK activity in a mouse hypothalamic glucose-sensing cell line (GT1-7) and mouse hypothalamic ex vivo slices. Perforated-patch electrophysiological recordings of GT1-7 cells investigated the acute and chronic effects of IL-6 (5ng/ml and 20ng/ml) on acute hypoglycemia (0.5mM glucose)-induced membrane hyperpolarization. Acute exposure of GT1-7 cells to 5ng/ml and 20ng/ml IL-6 had no significant hyperpolarizing effect during hypoglycemia (0.5mM). Contrastingly, antecedent 3 hour exposure to 5ng/ml IL-6 followed by a 24 hour recovery period significantly attenuated the response of GT1-7 cells to 0.5mM (ΔVControl = 13.4 ± 1.3mV vs ΔVIL6 = 8.6 ± 2.0mV, p<0.05; n=10-13). Additionally, IL-6 was found to significantly increase acute AMPK activity in mouse VMH ex vivo slices and GT1-7 cells, as demonstrated by increased AMPK phosphorylation, increased phosphorylation of ACC and direct AMPK activity assay compared to controls. Moreover, antecedent 3 hour exposure of GT1-7 cells to 5ng/ml IL-6 attenuated subsequent hypoglycemia-induced AMPK phosphorylation. Thus, prior exposure of hypothalamic GT1-7 cells and VMH ex vivo slices to IL-6 reduced their responsiveness to a subsequent hypoglycemia challenge. We conclude that rises in systemic or local IL-6 may contribute, at least in part, to hypoglycemia-induced suppression of hypothalamic glucose-sensing.