N-stearoyltyrosine (NsTyr), a synthesized anandamide (AEA) analogue, could exert potent neuroprotective effects on cerebral ischemia models both in vivo and in vitro via intervening in multiple injuries. Glutamate, a major excitatory neurotransmitter, plays a critical role during stroke/cerebral ischemia. In this study, we explored the protective effects of NsTyr on glutamate neurotoxicity in PC12 cells and investigated its underlying mechanisms. NsTyr treatment attenuated glutamate-induced oxidative toxicity in a dose-dependent manner and the best performance was observed at 10 μΜ. NsTyr treatment suppressed glutamate-induced upregulation of lipoxygenase 12/15 (LOX 12/15) activity and reactive oxygen species (ROS) elevation, attenuated the increase of BH3-interacting domain death agonist (Bid) in the mitochondria, prevented the loss of mitochondria membrane potential and consequently inhibited apoptosis-inducing factor (AIF) translocation into the nucleus. The results demonstrated that NsTyr could protect cells against AIF-mediated caspase-independent cell death induced by glutamate, which may be due to the blockage of Bid-mediated mitochondrial damage via reducing LOX 12/15 activity and ROS accumulation.