We previously reported 12 antioxidative flavonoids isolated from the n-BuOH extract of Inula britannica (Asteraceae). This prompted us to investigate further whether these flavonoids also showed antioxidative activity upon live cells grown in a culture system. Among the 12 flavonoids tested, only patuletin, nepetin, and axillarin protected primary cultures of rat cortical cells from oxidative stress induced by glutamate. These flavonoids exerted significant neuroprotective activity when they were administered either before or after the glutamate insult. Treatment with these flavonoids maintained the activities of such antioxidant enzymes as catalase, glutathione-peroxidase, and glutathione reductase, all of which play important roles in the antioxidative defense mechanism. Moreover, these three flavonoids also attenuated significant drops in glutathione induced by glutamate which is a routine concomitant of oxidative stress by inhibiting glutathione diminution. Accordingly, these flavonoids did not stimulate the synthesis of glutathione. With regard to structure-activity relationships, our results indicated that the 6-methoxyl group in the A ring and the 3', 4'-hydroxyl groups in the B ring are crucial for the protection against the oxidative stress; glycosylation greatly reduced their protective activities. Collectively, these results indicated that patuletin, nepetin, and axillarin strongly protect primary cultured neurons against glutamate-induced oxidative stress.