In Alzheimer's disease (AD), the accumulation of senile plaques composed of neurotoxic amyloid β (Aβ) is known to be one of the causes. Shati/Nat8l, a gene related to neuropsychiatric disorders, encodes an enzyme that biosynthesizes N-acetyl aspartate (NAA) from aspartate and acetyl CoA. Studies on AD patients and model mice show that NAA and Shati/Nat8l are associated with AD pathology. We previously demonstrated that hippocampal overexpression of Shati/Nat8l in 5xFAD mice, an AD model, improved cognitive suppress without altering the number or size of Aβ plaques. To investigate the cellular mechanisms underlying the neuroprotective effects of Shati/Nat8l on Aβ neurotoxicity, we constructed a vector containing the full-length Shati/Nat8l sequence and transfected it into Neuro-2a cells to produce a stably Shati/Nat8l-overexpressing cell line (N2A-Shati). N2A-Shati cells expressed threefold higher Shati/Nat8l mRNA levels compared with a control cell line (N2A-Control). Treatment with Aβ for 48 h reduced the viability of N2A-Shati and N2A-Control cells at concentrations ≧ 0.03 μM compared to their own vehicle. Exposure to 0.03 μM Aβ for 24 h did not induce any detectable changes in mitochondrial mass or mitochondrial membrane potential in either N2A-Control or N2A-Shati cells. However, N2A-Shati cells demonstrated reduced pyruvate dehydrogenase kinase 1 (Pdk1) mRNA expression and enhanced nuclear respiratory factor 1 (Nrf1) and mitochondrial transcription factor A (Tfam) mRNA expression levels. These results suggest that, although Shati/Nat8l does not significantly affect cell viability, mitochondrial mass, or membrane potential, it could modulate specific intracellular pathways.