Alzheimer’s disease (AD) is characterized by amyloid plaques and neurofibrillary tangles accompanied with progressive neurite loss. Mitochondria play pivotal roles in Alzheimer’s disease development. PRDX3 is a mitochondrial peroxide reductase responsible for H2O2 scavenge and signaling transduction. In this study, we found knockdown PRDX3 in N2a-APPSwe cell line induced neurite outgrowth, but not reduced cell viability against tert-butyl hydroperoxide (TBHP) induced cell damage. We found knockdown of PRDX3 induced dysregulation of one hundred proteins by Tandem Mass Tag (TMT) labeled proteomic study. Gene Ontology analysis revealed that the dysregulated proteins are enriched on establishment of protein localization to plasma membrane, lipid catabolic process, and intermediate filament cytoskeleton organization. String analysis showed closely protein-protein interactions among the dysregulated proteins. Annexin A1 (ANXA1), serine/threonine-protein phosphatase 2A catalytic subunit alpha isoform (PP2A) and glutathione S-transferase Mu 2 (GSTM2) were significantly upregulated in PRDX3 knockdown N2a-APPswe cell lines and verified by western blot. Our study for the first time revealed PRDX3 may also played important roles on neurite outgrowth and related AD development.