It is widely recognized that glutamate (Glu)-induced cytotoxicity, intracellular calcium overload and the excessive free radical production are key events in the development and progression of ischemic brain injury. dl-3-n-butylphthalide (NBP), an anti-ischemic agent, has therapeutic effects in animal models of vascular dementia. The aim of the present study was to investigate the protective effect of 3-butyl-6-fluoro-1(3H)-isobenzofuranone (6-F-NBP), a derivative of NBP on Glu-induced cytotoxicity in rat pheochromocytoma (PC12) cells, and to compare this action with NBP. The results showed that after 24-h incubation with Glu (5 mM), cell viability and mitochondrial membrane potential (MMP) were decreased. In contrast, the content of reactive oxygen species (ROS), activity of nitric oxide synthase (NOS), and apoptosis rate, as well as intracellular accumulation of [Ca2+]i, were increased, 6-F-NBP inhibited the damage induced by Glu in a dose-dependent manner and exerted a more potent effect than NBP, indicating that 6-F-NBP exhibited a protective effect against Glu-induced cytotoxicity in cultured PC12 cells. Drug Dev Res 73: 11–17, 2012. © 2011 Wiley Periodicals, Inc.