Some endocannabinoids have been known to express anti-inflammatory and antioxidant actions independent of cannabinoid receptors. In this respect, we investigated whether N-acyl 5-hydroxytryptamines (5-HTs) might prevent against glutamate-induced oxidative cytotoxicity in HT-22 cells, and attempted to elucidate the mechanism for their cytoprotective action. N-acyl 5-HTs with palmitoyl, stearoyl, arachidonoyl or docosahexaenoyl chain expressed a remarkable protective effect on glutamate-induced cytotoxicity. Additionally, glutamate-induced oxidative stress, represented by the increase of reactive oxygen species level and the reduction of glutathione level, was prevented markedly by N-acyl 5-HTs at submicromolar levels. Further, N-palmitoyl 5-HT, the most cytoprotective, enhanced antioxidant defense by up-regulating the expression of antioxidant enzymes such as heme oxygenase-1, glutamate-cysteine ligase catalytic subunit or NAD(P)H quinine oxidoreductase-1 in the presence or absence of glutamate. Consistent with this, N-palmitoyl 5-HT stimulated nuclear translocation of Nrf2 in early phase (2 h), and this effect was remarkably suppressed by inhibitors of PI3K, PDK-1, Akt or p38 MAPK. Additionally, N-palmitoyl 5-HT suppressed glutamate-induced activation of ERK in late phase (12 h), but not in early phase (2 h), presumably supporting the implication of MEK/ERK pathway in glutamate-induced cytotoxicity. Collectively, it is suggested that N-acyl 5-HTs may attenuate glutamate-induced cytotoxicity via the activation of PI3K/PDK-1/Akt- and p38 MAPK-dependent Nrf2 signaling in early phase as well as the suppression of MEK/ERK pathway in late phase.