Alzheimer disease (AD), a progressive neurodegenerative disorder, is characterized by cognitive decline and the accumulation of senile plaques in the brain. Amyloid β protein (Aβ) in the plaques is thought to be responsible for the memory loss in AD patients. [Gly14]-humanin (HNG), a derivative of humanin (HN), has much stronger neuroprotective effects than natural HN in vitro. However, clarification of the Aβ active center and the neuroprotective mechanism of HN still need in vivo evidence. The present study first compared the in vivo biological effects of three Aβ fragments (1–42, 31–35, and 35–31) on spatial memory in rats, and investigated the neuroprotective effects and molecular mechanisms of HNG. The results showed that intrahippocampal injection of Aβ_1–42 and Aβ_31–35 almost equally impaired spatial learning and memory, but the reversed sequence Aβ_35–31 did not have any effect; a high dose of Aβ_31–35 (20 nmol) produced a more detrimental response than a low dose (2 nmol); Aβ_31–35 injection also disrupted gene and protein expression in the hippocampus, with up-regulation of caspase3 and down-regulation of STAT3; pretreatment with HNG not only protected spatial memory but also rescued STAT3 from Aβ-induced disruption; and the neuroprotective effects of HNG were effectively counteracted by genistein, a specific tyrosine kinase inhibitor. These results clearly show that sequence 31–35 in Aβ is the shortest active center responsible for the neurotoxicity of Aβ from molecule to behavior; and HNG protects spatial learning and memory in rats against Aβ-induced insults; and probably involves the activation of tyrosine kinases and subsequent beneficial modulation of STAT3 and caspase3.