Excitotoxicity, the overstimulation of ionotropic glutamate receptors is a key process of neuronal loss in acute ischaemic stroke and chronic neurological disorders. However, exactly how neurons die in excitotoxicity still remains unclear. Using stable-isotope dimethyl labelling based quantitative proteomic and phosphoproteomic approaches, we identified at least 80 neuronal proteins showing perturbed expression and 59 phosphoproteins showing significant changes in phosphorylation following 15 min and 4 h of glutamate-induced excitotoxicity. Most of the identified neuronal proteins exhibited reduced expression in excitotoxicity. Signalling network analysis using IPA with these identified protein molecules revealed (i) 14-3-3-mediated signalling, (ii) remodelling of epithelial adherens junctions, (iii) cell cycle including G2/M DNA damage checkpoint regulation, (iv) Myc-mediated apoptosis signalling, (v) PI3K/Akt signalling and (vi) Erk/MAPK signalling as top dysregulated canonical pathways in excitotoxicity. Using similar approach, we also identified significantly dysregulated neuronal proteins and phosphoproteins that are downstream of neurotoxic GluN2B-containing extrasynaptic NMDA receptors. Representative proteomic data were validated by Western blot analysis, also changes in phosphorylation of Mef2c (Ser-222), Mff (Ser-146), Mlf2 (Ser-237) and Stmn1 (Ser-38) were validated by label-free full-scan precursor ions (MS1) quantitation analysis using isotopically labelled synthetic phosphopeptide standards. Our results collectively indicate that inactivation of a number of pro-survival signalling pathways and activation of a series of pro-death signalling pathways cooperate to cause neuronal demise in excitotoxicity. In summary, our findings shed light on the molecular mechanism of excitotoxic neuronal death and identified neuronal proteins are potential targets for the development of neuroprotectants to reduce excitotoxic brain damage in neurological disorders.