β-Hydroxybutyric acid (BHBA) acts in the brain to influence feeding behaviour, but the underlying molecular mechanisms are unclear. GT1-7 hypothalamic cells expressing orexigenic agouti-related peptide (AGRP) were used to study the AMP-activated protein kinase (AMPK) pathway known to integrate dietary and hormonal signals for food intake regulation. In a 25 mM glucose culture medium, BHBA increased intracellular calcium concentrations and the expression of monocarboxylate transporter 1 (MCT1 (SLC16A1)). Phosphorylation of AMPK-α (PRKAA1 and PRKAA2) at Thr(172) was diminished after 2 h but increased after 4 h. Its downstream target, the mammalian target of rapamycin, was increasingly phosphorylated on Ser(2448) after 2 h but not changed after 4 h of BHBA treatment. After 4 h, BHBA treatment also increased Agrp mRNA expression. This increase was prevented by preincubation with the AMPK inhibitor Compound C. The inhibition of MCT1 activity by p-hydroxymercuribenzoate suppressed BHBA-stimulated AMPK phosphorylation but did not prevent BHBA-induced Agrp mRNA expression. This finding demonstrates that BHBA triggers the AMPK pathway resulting in orexigenic signalling under 25 mM glucose culture conditions. Under conditions of 5.5 mM glucose, however, BHBA marginally increased intracellular calcium but significantly decreased AMPK phosphorylation and Agrp mRNA expression, demonstrating that under physiological conditions BHBA reduces central orexigenic signalling.