Although the brain utilizes glucose for energy production, individual brain cells may to some extent utilize substrates derived from glucose. Thus, it has been suggested that neurons consume extracellular lactate during synaptic activity. However, the precise role of lactate for fuelling neuronal activity is still poorly understood. Recently, we demonstrated that glucose metabolism is up‐regulated in cultured glutamatergic neurons during neurotransmission whereas that of lactate is not. Here, we show that utilization of glucose but not lactate correlates with NMDA‐induced neurotransmitter glutamate release in cultured cerebellar neurons from mice. Pulses of NMDA at 30, 100, and 300 μM, leading to a progressive increase in both cytosolic [Ca²⁺] and release of glutamate, increased uptake and metabolism of glucose but not that of lactate as evidenced by mass spectrometric measurement of ¹³C incorporation into intracellular glutamate. In this manuscript, a cascade of events for the preferential neuronal utilization of glucose during neurotransmission is suggested and discussed in relation to our current understanding of neuronal energy metabolism.