The accumulation of free fatty acids induces lipotoxicity in neural cells. Estrogen, 17β-estradiol (E2) protects against the damage of cells in various organs and tissues. However, the role of E2 on lipotoxicity in neural cells remains unclear. In this study, we investigated the effects of E2 on stearic acid (saturated fatty acid)- and oleic acid (unsaturated fatty acid)-induced cytotoxicity in retinoic acid-induced mouse neuroblastoma Neuro-2a differentiated into neural cells. Cell viability was evaluated by lactate dehydrogenase release from Neuro-2a neural cells. Stearic acid and oleic acids suppressed the cell viability in a dose-dependent manner. E2 prevented oleic acid-induced cytotoxicity but had no effect on stearic acid-induced cytotoxicity. ERα-selective agonist prevented cytotoxicity in Neuro-2a neural cells. In contrast, ERβ-selective agonist slightly significantly enhanced the cytotoxicity in the presence of oleic acid. Oleic acid, but not stearic acid, increased the mRNA level of p62/Sqstm1. E2 treatment statistically significantly, but slightly, enhanced the stearic acid-induced Bax expression. In contrast, E2 and ERα-selective agonist inhibited the oleic acid-induced the p62/Sqstm1 expression. Our results suggested that fatty acids induced cytotoxicity in Neuro-2a neural cells, and estrogen prevented the oleic acid-induced cytotoxicity via ERα, but not ERβ. Further studies are needed to understand the role of ERβ in neuron injury under normal conditions.