Malathion is an organophosphate with severe neurotoxic effects. Upon acute exposure, malathion initially enhances cholinergic activity by inhibition of acetylcholinesterase, which is its major pathological mechanism. Malathion also induces non-cholinergic neuronal cell death in neurodegenerative conditions; the associated molecular mechanism is not well-characterized. To investigate the molecular mechanism of malathion-induced cell death, N2a mouse neuroblastoma cells were exposed to malathion and cell death-related parameters were examined. Malathion reduced cell viability mainly by apoptosis through mitochondrial dysfunction in N2a cells, as judged by an increase in the level of the pro-apoptotic protein Bax and decrease in the levels of the anti-apoptotic proteins p-Akt and Bcl2, resulting in cytochrome c release and caspase-dependent DNA fragmentation and condensation. Malathion treatment also induced autophagy and lysosomal membrane permeabilization (LMP) in N2a cells. LMP caused a lessening of autophagic flux via inhibition of lysosomal fusion with the autophagosome. LMP-induced cathepsin B release and its proteolytic effect may intensify apoptotic insults. Moreover, malathion-exposed N2a cells showed a marked reduction in the levels of the neuronal marker proteins vascular endothelial growth factor and heart fatty acid binding protein 3, along with diminished neuritogenesis in N2a cells and nerve growth factor secretion in C6 glioma cells. Our data suggest that the non-cholinergic effect of malathion may be mediated by apoptotic cell death via LMP induction in N2a cells. Malathion-treated N2a cells can be utilized as an in vitro model system to screen natural and new chemical drug candidates for neurodegenerative diseases such as Alzheimer's disease.