Glutamate-induced excitotoxicity is a major contributor to motor neuron degeneration in the pathogenesis of amyotrophic lateral sclerosis (ALS). The spinal cord × Neuroblastoma hybrid cell line (NSC-34) is often used as a bona fide cellular model to investigate the physiopathological mechanisms of ALS. However, the physiological response of NSC-34 to glutamate remains insufficiently described. In this study, we evaluated the relevance of differentiated NSC-34 (NSC-34D) as an in vitro model for glutamate excitotoxicity studies. NSC-34D showed morphological and physiological properties of motor neuron-like cells and expressed glutamate receptor subunits GluA1-4, GluN1 and GluN2A/D. Despite these diverse characteristics, no specific effect of glutamate was observed on cultured NSC-34D survival and morphology, in contrast to what has been described in primary culture of motor neurons (MN). Moreover, a small non sustained increase in the concentration of intracellular calcium was observed in NSC-34D after exposure to glutamate compared to primary MN. Our findings, together with the inability to obtain cultures containing only differentiated cells, suggest that the motor neuron-like NSC-34 cell line is not a suitable in vitro model to study glutamate-induced excitotoxicity. We suggest that the use of primary cultures of MN is more suitable than NSC-34 cell line to explore the pathogenesis of glutamate-mediated excitotoxicity at the cellular level in ALS and other motor neuron diseases.