Although several in vitro and ex vivo evidence support the existence of lactate exchange between astrocytes and neurons, a direct demonstration in vivo is still lacking. In the present study, a lentiviral vector carrying a short hairpin RNA (shRNA) was used to downregulate the expression of the monocarboxylate transporter type 2 (MCT2) in neurons of the rat somatosensory cortex (called S1BF) by ~ 25%. After one hour of whisker stimulation, HRMAS 1H-NMR spectroscopy analysis of S1BF perchloric acid extracts showed that while an increase in lactate content is observed in both uninjected and shRNA-control injected extracts, such an effect was abrogated in shMCT2 injected rats. A 13C-incorporation analysis following [1-13C]glucose infusion during the stimulation confirmed that the elevated lactate observed during activation originates from newly synthesized [3-13C]lactate, with blood-derived [1-13C]glucose being the precursor. Moreover, the analysis of the 13C-labeling of glutamate in position C3 and C4 indicates that upon activation, there is an increase in TCA cycle velocity for control rats while a decrease is observed for MCT2 knockdown animals. Using in vivo localized 1H-NMR spectroscopy, an increase in lactate levels is observed in the S1BF area upon whisker stimulation for shRNA-control injected rats but not for MCT2 knockdown animals. Finally, while a robust BOLD fMRI response was evidenced in control rats, it was absent in MCT2 knockdown rats. These data not only demonstrate that glucose-derived lactate is locally produced following neuronal activation but also suggest that its use by neurons via MCT2 is probably essential to maintain synaptic activity within the barrel cortex.