Voltage-gated sodium channel beta 2 (Nav beta 2), as an unconventional substrate of beta-site amyloid precursor protein cleaving enzyme 1, is involved in regulating the neuronal surface expression of sodium channels. A previous study demonstrated that knockdown of Nav beta 2 protected neurons and induced spatial cognition improvement by partially reducing pathological amyloidogenic processing of amyloid precursor protein (APP) in aged APP/presenilin 1 (PS1) transgenic mice. The present study aimed to investigate whether Nav beta 2 knockdown altered APP metabolism via regulation of the A beta-degrading enzyme neprilysin (NEP). APPswe/PS1 Delta E9 mice (APP/PS1 transgenic mice with a C57BL/6J genetic background) carrying a Nav beta 2-knockdown mutation (APP/PS1/Nav beta 2-kd) or without Nav beta 2 knockdown (APP/PS1) were used for cell culture and further analysis. The present results demonstrated that in APP/PS1 mouse-derived neurons, Nav beta 2 knockdown partially reversed the reduction in pathological APP cleavage, and the recovery of neurite extension and neuron area. Additionally, Nav beta 2 knockdown increased NEP activity and levels, and the levels of intracellular domain fragment binding to the NEP promoter. The present findings suggested that knockdown of Nav beta 2 reversed the APP/PS1 mutation-induced deficiency in amyloid beta degradation by regulating NEP.