Oxidative stress is involved in many neurological diseases, including Alzheimer's disease. Punicalagin (PC) is a hydrolysable polyphenol derived from Punica granatum and a potent antioxidant. In this study, the neuroprotective effect of PC on glutamate-induced oxidative stress was evaluated in the mouse hippocampal cell line, HT22. PC treatment protected HT22 cells from glutamate-induced cell death in a concentration-dependent manner, potentially attenuated glutamate-induced intracellular reactive oxygen species (ROS) and restored the mitochondrial membrane depolarization. Metabolic alterations after glutamate-induced oxidative stress and the protective effect of PC were evaluated with HPLC and GC-MS profiling methods with multivariate statistical analyses. Alterations in ten metabolites were identified, including amino acids, aspartic acid, asparagine, threonine, anserine, cysteine, tryptophan, lysine, as well as fatty acids palmitic acid, stearic acid, and palmitoleic acid. Metabolic pathway analysis revealed the involvement of multiple affected pathways, such as cysteine and methionine metabolism, tryptophan metabolism, alanine, aspartate, and glutamate and fatty acid oxidation. These results clearly demonstrate that PC is a promising therapeutic agent for oxidative stress-associated diseases.