Hyperpolarized [1-¹³C]pyruvate magnetic resonance (MR) spectroscopy has the unique ability to detect real-time metabolic changes in vivo owing to its high sensitivity compared with thermal MR and high specificity compared with other metabolic imaging methods. The aim of this study was to explore the potential of hyperpolarized MR spectroscopy for quantification of liver pyruvate metabolism during a hyperinsulinemic–isoglycemic clamp in mice. Hyperpolarized [1-¹³C]pyruvate was used for in vivo MR spectroscopy of liver pyruvate metabolism in mice. Mice were divided into two groups: (i) non-stimulated 5-h fasted mice and (ii) hyperinsulinemic-isoglycemic clamped mice. During clamp conditions, insulin and donor blood were administered at a constant rate, whereas glucose was infused to maintain isoglycemia. When steady state was reached, insulin-stimulated mice were rapidly infused with hyperpolarized [1-¹³C]pyruvate for real-time tracking of the dynamic distribution of metabolic derivatives from pyruvate, such as [1-¹³C]lactate, [1-¹³C]alanine and [¹³C]bicarbonate. Isotopomer analysis of plasma glucose confirmed ¹³C-incorporation from [1-¹³C]pyruvate into glucose was increased in fasted mice compared with insulin-stimulated mice, demonstrating an increased gluconeogenesis in fasted mice. The AUC ratios for [1-¹³C]alanine/[1-¹³C]pyruvate (38.2%), [1-¹³C]lactate/[1-¹³C]pyruvate (41.8%) and [¹³C]bicarbonate/[1-¹³C]pyruvate (169%) all increased significantly during insulin stimulation. Hyperpolarized [1-¹³C]pyruvate can be used for in vivo MR spectroscopy of liver pyruvate metabolism during hyperinsulinemic-isoglycemic clamp conditions. Under these conditions, insulin decreased gluconeogenesis and increased [1-¹³C]alanine, [1-¹³C]lactate and [¹³C]bicarbonate after a [1-¹³C]pyruvate bolus. This application of in vivo spectroscopy has the potential to identify impairments in specific metabolic pathways in the liver associated with obesity, insulin resistance and nonalcoholic fatty liver disease.