Although glucose is the primary cerebral fuel, the brain is able to metabolize other substrates in hypoglycemia. Nevertheless, the metabolic consequences of this pathology at the cellular level remain largely unknown. Taking advantage of the metabolic flux analysis (MFA) methodology, this work was aimed at investigating and quantifying the effects of hypoglycemia on cerebellar neurons. After 12 hr without glucose, primary cultures were incubated with medium containing [1,6-13C]glucose and unlabeled glutamine, and metabolism was monitored for 30 hr. Metabolic rates of glucose, lactate, and amino acids were determined based on cell supernatant analysis and used to estimate metabolic fluxes with MFA. Percent 13C enrichment time profiles of different keto and amino acids were measured by mass spectrometry in cell extracts and compared with the MFA results. Hypoglycemia decreased the glucose uptake rate and glycolytic metabolism by 35% whereas glutamine uptake was increased fourfold. Flux estimations fit well with data from 13C labeling dynamics, indicating a significant activation of the pyruvate recycling (PR) pathway, accounting for 43% of the total pyruvate synthesized under control conditions and up to 71% after hypoglycemia. Increased PR appeared to be due mainly to increased glutamine oxidation given the higher label dilution observed in the hypoglycemia group. In summary, this work provides new evidence for PR as an important pathway for glutamine oxidation in cerebellar neurons, particularly after glucose deprivation. © 2011 Wiley-Liss, Inc.