Metabolic imaging using hyperpolarized magnetic resonance can increase the sensitivity of MRI, though its ability to inform on relevant changes to biochemistry in humans remains unclear. In this work, we image pyruvate metabolism in patients, assessing the reproducibility of delivery and conversion in the setting of primary prostate cancer. We show that the time to max of pyruvate does not vary significantly within patients undergoing two separate injections or across patients. Furthermore, we show that lactate increases with Gleason grade. RNA sequencing data demonstrate a significant increase in the predominant pyruvate uptake transporter, monocarboxylate transporter 1. Increased protein expression was also observed in regions of high lactate signal, implicating it as the driver of lactate signal in vivo. Targeted DNA sequencing for actionable mutations revealed the highest lactate occurred in patients with PTEN loss. This work identifies a potential link between actionable genomic alterations and metabolic information derived from hyperpolarized pyruvate MRI. Display omitted •HP pyruvate can be safely infused multiple times and measures reproducible kinetics•Tumors with increased Gleason grades had increased levels of hyperpolarized lactate•Regions of high HP lactate correlated with elevated monocarboxylate transporter 1•Conversion of HP pyruvate to lactate was increased in tumors with loss of PTEN Non-invasive tools are needed to reveal metabolic phenomena in humans. In this translational study, Granlund et al. utilize metabolic imaging to interrogate prostate cancer, finding a mechanistic link between increased metabolism and tumor grade. Thus, real-time metabolic imaging in humans can not only provide clinically relevant tools but also uncover new biology.