Hyperpolarized [1- ¹³ C]pyruvate enables direct in vivo assessment of real-time liver enzymatic activities by ¹³ C magnetic resonance. However, the technique usually requires the injection of a highly supraphysiological dose of pyruvate. We herein demonstrate that liver metabolism can be measured in vivo with hyperpolarized [1- ¹³ C]pyruvate administered at two- to three-fold the basal plasma concentration. The flux through pyruvate dehydrogenase, assessed by ¹³ C-labeling of bicarbonate in the fed condition, was found to be saturated or partially inhibited by supraphysiological doses of hyperpolarized [1- ¹³ C]pyruvate. The [ ¹³ C]bicarbonate signal detected in the liver of fasted rats nearly vanished after treatment with a phosphoenolpyruvate carboxykinase (PEPCK) inhibitor, indicating that the signal originates from the flux through PEPCK. In addition, the normalized [ ¹³ C]bicarbonate signal in fasted untreated animals is dose independent across a 10-fold range, highlighting that PEPCK and pyruvate carboxylase are not saturated and that hepatic gluconeogenesis can be directly probed in vivo with hyperpolarized [1- ¹³ C]pyruvate.