Agouti-related peptide (AgRP) neurons in the arcuate nucleus of the hypothalamus are essential for integrating hormonal and nutrient cues conveying negative energy balance. In support of this, ablation of AgRP neurons in adulthood results in starvation. Although AgRP neurons increase food intake, motivation for food, hepatic glucose production and decrease energy expenditure, the molecular mechanisms responsible for sensing negative energy balance remain unknown. We reasoned mitochondrial mechanisms regulating energy metabolism acts as metabolic sensors that influence AgRP function. Thus, we deleted carnitine acyltransferase (Crat), a mitochondrial matrix enzyme that regulates mitochondrial carnitine metabolism, from AgRP neurons and examined the effect on energy metabolism during negative energy balance. Cre-dependent reporter expression using loxSTOPlox tdTomato mice revealed that the majority of AgRP neurons expressed tdTomato and fluorescent-activated cell sorting indicated successful deletion of Crat mRNA transcripts from AgRP neurons. We show that selective deletion of Crat in AgRP neurons decreases food intake after fasting, alters feeding behaviour, as assessed by automatic feeding cages (BioDAQ, Research Diets, USA), and decreases the motivation to obtain food during fasting. After fasting, AgRP Crat KO also had lower concentrations of liver glycogen relative to WT mice, suggesting impaired hepatic glucose metabolism. A finding that was paralled with qPCR analysis of genes involved in hepatic glucose production. We also observed increased liver triglyceride levels and decreased plasma triglyceride levels, after fasting in KO mice compared to WT littermates. These findings suggest that Crat in AgRP neurons is involved in the sensing negative energy balance and engaging neural circuits to regulate feeding behaviour and liver function.