Ninjinyoeito (NYT), a Japanese herbal medicine, is widely used to treat patients with insomnia, anemia, amnesia, and neurosis. Recently, NYT was reported to be clinically effective in Alzheimer's disease (AD). To address the mechanism underlying effect of NYT on AD, we examined the effects of NYT in β-amyloid (Aβ)_25–35-exposed primary cocultured astrocytes and neurons. The effects of NYT on neurotoxicity induced by Aβ_25–35 were assessed by immunocytochemical assays and Sholl analysis of microtubule-associated protein 2 (MAP2)-positive and tau-positive neurites. Aβ_25–35 treatment attenuated the arborization of axons and dendrites of single autaptic hippocampal neurons in a concentration-dependent manner. NYT treatment ameliorated the Aβ_25–35-induced impairment of tau-positive axon outgrowth. However, NYT did not ameliorate the Aβ_25–35-induced suppression of MAP2-positive dendrite arborization. RT-PCR analysis demonstrated that NYT increases the expression of nerve growth factor (Ngf) but not brain derived neurotrophic factor (Bdnf) in Aβ_25–35-exposed primary cocultured astrocytes and neurons. Our results indicate that NYT protects against Aβ_25–35-induced neuronal injury through induction of nerve growth factor expression. These findings provide a mechanistic basis for treating AD with NYT.