Parkinson's disease (PD) is a common chronic neurodegenerative disorder associated with aging that primarily caused by the death of dopaminergic neurons in the substantia nigra pars compacta (SN). Retinoic acid (RA)-differentiated human neuroblastoma SH-SY5Y cells (SH-SY5Y+RA) have been broadly utilized in studies of mechanisms of the pathogenesis underlying 1-Methyl-4-phenyl pyridinium (MPP(+))-induced PD models. Here, we investigated the neuroprotective mechanisms of insulin on MPP(+)-induced neurotoxicity on SH-SY5Y+RA cells. Recent studies suggest that insulin has a protective effect against oxidative stress but not been elucidated for PD. In this study, pretreatment of insulin prevented the cell death in a dose dependent manner and lowered nitric oxide (NO) release, reactive oxygen species (ROS), and calcium ion (Ca(2+)) influx induced by MPP(+). Insulin also elevated tyrosine hydroxylase (TH) and insulin signaling pathways in dopaminergic neuron through activating PI3K/Akt/GSK-3 survival pathways which in turn inhibits MPP(+)-induced iNOS and ERK activation, and Bax to Bcl-2 ratio. These results suggest that insulin has a protective effect on MPP(+)-neurotoxicity in SH-SY5Y+RA cells.