Deuterium Metabolic Imaging (DMI) is a novel approach providing high 3D spatial resolution metabolic data from both animal models and human subjects. DMI relies on 2H MRSI in combination with administration of 2H-labeled substrates. In a rat glioma model we show how DMI combined with administration of [6,6’-2H2]-glucose and [2H3]-acetate allowed spatial mapping of differences in metabolism between normal brain and glioma. DMI data after infusion of 2H-labeled glucose revealed high glucose uptake and lactate production but limited glucose oxidation in glioma. Furthermore, DMI revealed acute therapeutic effects of a drug (dichloroacetate) targeting glucose oxidation.