Multidrug resistance associated protein 5 (MRP5/ABCC5) is a drug efflux transporter, and its gene expression in murine primary cultured neurons is much higher than other ABCC family members although its physiological functions are largely unknown. MRP5 also pumps out the neurotransmitter N-acetylaspartyl-glutamate (NAAG), which suppresses excitatory neurotoxicity in the neurodegenerative diseases. We therefore hypothesized that MRP5 may play a protective role in the brain by regulating NAAG transport. In this study, the effect of Mrp5 knockdown on neurotoxicity was investigated. Adeno-associated virus-PHP.eB carrying shRNA targeting Mrp5 (AAV-shMrp5) or LacZ (AAV-shLacZ, negative control) expressing fluorescent protein Zsgreen1 were first injected into the cerebellum in mice, and cerebellar sections were observed under a fluorescence microscope after 12 days, showing that the number of calbindin-positive neurons in AAV-shMrp5-treated group was lower than that in negative control. The neuronal cell line Neuro2A was next transfected with siRNA targeted to Mrp5 (siMrp5), showing a lower expression of Mrp5 and a higher intracellular NAAG concentration compared to negative control siRNA. Furthermore, the cell viability measured by MTT assay and cytotoxicity evaluated by LDH assay in the siMrp5-transfected group were lower and higher, respectively, than the control group whereas NAAG treatment recovered the siMrp5-induced decrease in cell viability. These results suggest that Mrp5 may be involved in neuroprotection by regulating NAAG level.