This study was aimed to investigate pharmacological properties of notoginsenoside R1 (NG-R1), an ingredient in Panax notoginseng, against the neurotoxicity of glutamate (Glu) in primary cultured mouse neocortical neurons. Cerebral cortex was isolated from fetal mice at 15 days after gestation, followed by culture in DMEM in the presence of fetal calf serum (FCS) for the initial 3 days and subsequent culture in DMEM in the absence of FCS with medium change every 3 days. Brief exposure to Glu drastically reduced cell viability 24 h later in cortical neurons cultured for 7 days, while simultaneous addition of NG-R1 led to significant protection against the loss of cellular viability by Glu. NG-R1 inhibited the elevation of intracellular Ca2+ ions, the generation of intracellular reactive oxygen species (ROS) and the depolarization of mitochondrial membrane potential in cultured cortical neurons exposed to Glu. Glu decreased the expression of Bcl-2 and increased that of Bax in cultured cortical neurons, whereas further addition of NG-R1 resulted in significant protection against the Glu-induced changes in Bcl-2 and Bax expressions. These results suggest that particular notoginsenosides may protect neurons from the neurotoxicity of Glu through a mechanism related to the suppression of the elevation of intracellular Ca2+ ions, generation of intracellular ROS, depolarization of mitochondrial membrane potential and induction of apoptosis.