<p>Deuterium metabolic imaging (DMI) is a new imaging approach that provides unique, complementary information to anatomical MRI of brain tumors. Preclinical DMI studies have demonstrated excellent image contrast following intravenous infusion of deuterated choline (²H₉-Cho) at a severalfold higher dose than recommended for humans. We investigated DMI performance in rat glioblastoma models after oral administration of a ²H₉-Cho dose recommended for humans. DMI, following the three daily oral low doses, resulted in ²H₉-Cho concentrations in the tumor and tumor-to-normal-brain image contrast comparable to a single, high intravenous dose. Further, ²H and 2D ¹H-¹⁴N HSQC NMR on excised tumor tissue revealed that oral administration led to increased contributions from Cho-derived molecules that were products of tumor metabolism compared to intravenous infusion of ²H₉-Cho. These results can advance clinical translation of Cho-DMI as a noninvasive imaging tool for brain tumor characterization by demonstrating the feasibility of an oral intake approach using a clinical dose.</p>