4-Hydroxynonenal (4HNE) is a toxic aldehyde which can accumulate during neurodegenerative diseases, such as AD. 4HNE-induced neuronal cytotoxicity includes the damage of neurite growth as well as a potential threat leading to the neuronal cell death. This study was designed to examine whether overexpression of aldehyde dehydrogenase-2 (ALDH2) affects 4HNE-induced neurite outgrowth blockage and neuronal death in primary hippocampal neurons in vitro. Plasmid-encoding rat ALDH2 was constructed and transfected into cultured rat hippocampal neurons. In vitro-cultured rat hippocampal neurons with the transfection of ALDH2 gene were showing resistance to 4HNE-induced neurite damage. Overexpression of ALDH2 in cultured rat hippocampal neurons blocked 4HNE-induced (3.2 μM for 24 h) reduction of neurite outgrowth and branching. In addition to the effect on neurite growth, ALDH2 overexpression also can protect neurons from 4HNE-evoked (10 μM for 24 h) neuronal death. Furthermore, we found that overexpression of ALDH2 can decrease the caspase-3 protein expression level; at the same time, it can decrease the reactive oxygen species (ROS) level and the disruption of mitochondrial transmembrane potential in cultured hippocampal neurons. Our data suggested that overexpressed ALDH2 gene may moderate 4HNE-induced neuronal death by regulating caspase-3 protein and ROS level in cultured hippocampal neurons. Based on these findings, ALDH2 gene can be a potential therapeutic target for treatment of neurodegenerative diseases, such as AD.