Hyperpolarized [1-13C]pyruvate magnetic resonance spectroscopic imaging (MRSI) is a non-invasive metabolic imaging modality intended to inform the state of metabolic reprograming and carbon flux in tumors. Elevated hyperpolarized [1-13C]pyruvate-to-[1-13C]lactate conversion rates in aggressive tumors have been reported to be associated with enhanced glycolytic flux and lactate dehydrogenase A (LDHA) activity (Warburg effect). Herein, cross-sectional analysis using genetic and pharmacological tools in mechanistic studies applied to well-defined genetically-engineered cell lines and tumors demonstrated that initial hyperpolarized [1-13C]pyruvate-to-[1-13C]lactate conversion rates as well as global conversion were highly dependent on and critically rate-limited by the transmembrane influx of [1-13C]pyruvate mediated predominated by monocarboxylate transporter-1 (MCT1). Specifically, in a diffusion-unlimited cell-encapsulated alginate bead model, induced siRNA knockdown or overexpression of MCT1 quantitatively inhibited or enhanced, respectively, unidirectional pyruvate influxes and [1-13C]pyruvate-to-[1-13C]lactate conversion rates, independent of LDHA activity. Similarly, in tumor models in vivo, hyperpolarized [1-13C]pyruvate-to-[1-13C]lactate conversion was highly dependent on and critically rate-limited by the induced transmembrane influx of [1-13C]pyruvate mediated by MCT1. Furthermore, Kaplan–Meier survival analysis for patients with pancreatic, renal, lung and cervical cancers showed that high-level expression of MCT1 correlated with poor overall survival, and in selected tumors, MCT1 expression correlated with LDHA expression; thus, hyperpolarized [1-13C]pyruvate MRSI may provide a non-invasive functional assessment of MCT1 as a prognostic marker in select patient populations. Overall, hyperpolarized [1-13C]pyruvate MRSI primarily measures MCT1-mediated [1-13C]pyruvate transmembrane influx in vivo, not glycolytic flux or LDHA activity, driving a re-interpretation of this maturing new technology during clinical translation.