Alzheimer's disease (AD) is associated with damage to hippocampal neurons and declines in cognitive functions. The accumulation of amyloid peptides is regarded as a crucial event in the initiation of AD. The neurotoxicity induced by Aβ25–35 peptides was used to screen for cytoprotective factors in vitro, and the cognitive deficits induced by the injection of Aβ25–35 into the hippocampus were used to evaluate effect on learning and memory. Our previous study revealed that hydrolysate of polygalasaponins (HPS) clearly improve the cognitive deficits induced by the injection of Aβ25–35 in mice, but the potential active constituent of HPS remains unclear. The purposes of this study were to separate and purify the secondary saponins of HPS, screen for neuroprotective effects of the constituents in vitro, and to evaluate the effect of cognition in vivo. Various chromatographic methods were used to separate and purify the HPS. The neuroprotective effects were examined in Aβ25–35-damage-induced PC12 cells. The protective effect of tenuifolin on the cognitive impairments induced by Aβ25–35 injection was assessed using the Morris water maze and step-through passive avoidance tests. Tenuifolin and fallaxsaponin A were isolated from the HPS. Tenuifolin possessed neuroprotective effects against Aβ25–35-induced apoptosis in PC12 cells and significantly improved the cognitive deficits induced by the intrahippocampal injection of Aβ25–35 in mice. Thus, tenuifolin is one of the active constituents of HPS against the neurotoxicity induced by Aβ25–35 peptides in vitro and in vivo.