In our previous report, four limonoids, obacunone, limonin, fraxinellone, and calodendrolide, isolated from Dictamnus dasycarpus showed significant neuroprotective activity against glutamate toxicity in primary cultured rat cortical cells. This study investigated neuroprotective mechanism of these compounds using the same in vitro culture system. These four compounds showed significant neuroprotective activity at the concentration of 0.1 muM. They effectively inhibited calcium influx and overproduction of cellular nitric oxide and reactive oxygen species accompanied by glutamate-induced neurotoxicity. In addition, these compounds significantly preserved mitochondrial membrane potential and activities of antioxidative enzymes. Our results showed that obacunone, limonin, fraxinellone, and calodendrolide significantly protect primary culture cortical cells against glutamate-induced toxicity by preserving the antioxidant defense system. These compounds might offer potential drug development candidate for various neurodegenerative diseases involved with glutamate.