PurposeDetermine the feasibility of 1H‐[13C] MRS in the mouse hypothalamus using a 14.1T magnet.MethodsWe optimized the design of a 1H‐[13C] surface coil to maximize the signal‐to‐noise ratio of 1H‐[13C] MRS in the mouse hypothalamus. With enhanced signal, 13C accumulation in glucose metabolites was measured in a 8.7 µL voxel in the hypothalamus of 5 healthy mice during the continuous administration of [1,6‐13C2]glucose.ResultsAccumulation of 13C label in glucose C6 and lactate C3 was visible in the hypothalamus 11 min after glucose administration. The 13C fractional enrichment (FE) curves of lactate C3, glutamate and glutamine C4, glutamate+glutamine C3 and C2, GABA C2, C3, and C4, and aspartate C3 were measured with a time resolution of 11 min over 190 min. FE time‐courses and metabolic pool sizes were averaged to fit a novel one‐compartment model of brain energy metabolism that incorporates the main features of the hypothalamus.ConclusionDynamic 1H‐[13C] MRS is able to measure in vivo brain metabolism in small and deep areas of the mouse brain such as the hypothalamus, and it can be used to calculate metabolic fluxes, including glutamatergic and GABAergic metabolism as well as the contribution of metabolic sources other than glucose.