We have previously reported that a neuroprotective substance, serofendic acid, was purified and isolated from fetal calf serum. Here, we investigated the effect of serofendic acid on glutamate-induced apoptosis using rat primary cultures of cortical neurons. Exposure of the cortical cultures to relatively low concentration of glutamate (100 μM) induced neuronal death and nuclear fragmentation. Glutamate exposure also induced a transient increase in caspase-3 activity. A membrane-permeable inhibitor of caspase-3 (DEVD-CHO) prevented the glutamate neurotoxicity. Serofendic acid (0.01–10 μM) markedly prevented glutamate-induced apoptotic neuronal death and nuclear fragmentation. To elucidate the protective mechanism of serofendic acid, we first examined the effect on the glutamate-induced increase in intracellular Ca2+ concentration. Glutamate-induced increase in intracellular Ca2+ concentration was significantly inhibited by MK-801, a NMDA receptor antagonist, but not by serofendic acid. Next, we investigated the effect of serofendic acid on the loss of mitochondrial membrane potential induced by glutamate by using a fluorescence indicator, tetramethylrhodamine methyl ester (TMRM). Glutamate exposure resulted in a rapid reduction of TMRM fluorescence, indicating that mitochondrial membrane was depolarized by glutamate. Serofendic acid prevented the loss of mitochondrial membrane potential following glutamate exposure. Moreover, serofendic acid reduced the activation of caspase-3 induced by glutamate. Finally, serofendic acid directly inhibited the activity of recombinant human caspase-3, -7 and -8 at higher concentrations. These results indicate that serofendic acid prevents glutamate-induced apoptosis in cultured cortical neurons by the prevention of loss of mitochondrial membrane potential and the reduction of the process of caspase-3 activation.