Neurolathyrism is characterized by spastic paraparesis of the legs. It is caused by overconsumption of grass pea (Lathyrus sativus L.; Leguminosae). We studied toxicity of extracts of L. sativus seeds from two different areas—Bangladesh and Canada—toward rat primary neuron/glia culture. Both extracts showed acute neurotoxicity within 24 h when the 75% ethanol extracts were added to the neuron/glia culture. Fractionation of the extracts showed that the water-soluble fraction accounted for ca. 75–84% of total toxicity in which 3-N-oxalyl-l-2,3-diaminopropanoic acid (l-β-ODAP) was present at the highest concentration. Toxicity of the water-soluble fraction obtained from Bangladeshi seeds was significantly higher than that obtained from Canada. Effects of these fractions were reversed almost completely by 1,2,3,4-tetrahydro-6-nitro-2,3-dioxobenzo[f]quinoxaline-7-sulfonamide (NBQX), an antagonist of AMPA-receptor. They were partially reversed by group I metabotropic glutamate receptor antagonists (RS)-1-aminoindan-1,5-dicarboxylic acid, or (S)-α-methyl-4-carboxyphenyl-glycine [(S)-MCPG]. Nitric oxide synthase inhibitor NG-nitro-l-arginine methyl ester (l-NAME) strongly decreased the extracts’ toxicity. These data show that the neurotoxicity of grass pea seeds is attributable to l-β-ODAP, the toxicity of which is mediated by collective effects of l-β-ODAP on the AMPA-type receptor, metabotropic glutamate receptors, and NO production.