Glutamate is the major excitatory neurotransmitter in the central nervous system. Excessive glutamate is known to cause excitotoxicity. Umbelliferone is a coumarin derivative compound and has antioxidant, anti-inflammatory, and neuroprotective effects. Also, umbelliferone can show neuroprotective effect by crossing the blood-brain barrier. In our study, it was aimed to investigate the neuroprotective effect of umbelliferone on primary cortical neuron (PCN) culture. Umbelliferone was isolated from the roots of Ferulago cassia dichloromethane sub-extract. The cerebral cortex of newborn Sprague Dawley rats was used to obtain PCNs. To stimulate glutamate excitotoxicity, cells were exposed to 6x10(-5)M glutamate. Then different concentrations (10-1000 mu M) of umbelliferone were added into the medium and allowed to incubate for 24 and 72 h. MTT assay was used to measure cell viability. Total Antioxidant Status (TAS) and Total Oxidant Status (TOS) analyzes were used to evaluate reactive oxygen species. MTT results showed that cell viability was decreased with glutamate application. 25-250 mu M umbelliferone had a significant protective effect against glutamate excitotoxicity at 72 h (P<0.05). Consistent with MTT results, TAS analysis results showed 50-250 mu M umbelliferone increase the level of antioxidants in cells, which can help protect neurons against glutamate-induced excitotoxicity. In this study, umbelliferone showed a neuroprotective effect in PCN against glutamate excitotoxicity. These results suggest that umbelliferone may be used as therapeutic agent against glutamate excitotoxicity.