It is well established that obesity and diabetes may be influenced by defects in hypothalamic signalling, which are associated with high levels of free fatty acids (FFA) in the circulation. Hypothalamic arcuate (ARC) neurons are important in linking nutrient sensing with metabolism and feeding. Intracerebroventricular (icv) administration of the long-chain fatty acid, oleic acid (OA) and pharmacological manipulation of fatty acid metabolism alter energy balance and glucose homeostasis. The present study aims to elucidate the cellular mechanisms of OA on ARC neuron excitability of C57BL/6 mice and to determine whether this correlates with glucose-sensitivity. Whole-cell current-clamp recordings in the presence of 10 mM glucose revealed acute effects of OA (100 µM in the presence of 0.05% BSA) in 69% of ARC neurons of which 58% hyperpolarized and 11% depolarized. Similar outcomes were observed in perforated-patch recordings in the presence of 2 mM glucose with 32% hyperpolarizing and 26% depolarizing in response to OA. Our results show that the inhibitory effects of OA are exerted via activation of ATP-sensitive K (K_ATP) channels. Challenging perforated-patch recorded ARC neurons with a reduction in glucose from 2 mM to 0.1 mM showed that the majority of ARC neurons (92%; n = 23/25) displayed sensitivity to glucose, with 56% hyperpolarizing (glucose-excited (GE) neurons) and 36% depolarizing (glucose-inhibited (GI) neurons). Examination of identified glucose-sensing ARC neurons showed that GE neurons responded by activation of K_ATP and hyperpolarization, and GI neurons by depolarization respectively, in response to challenge with OA. Thus, our data indicate that the actions of OA on ARC neurons are glucose-independent as similar responses occur in different external glucose concentrations (10 and 2 mM) but that the response induced by OA correlates strongly with their glucose-sensing phenotype. Further studies are underway to determine whether glucose and oleic acid metabolism utilize similar intracellular mediators to elicit changes in ARC neuron excitability.