In early Alzheimer's disease (AD) brain, reduction of sigma-1 receptors (σ1R) is detected. In this study, we employed male heterozygous σ1R knockout (σ1R+/–) mice showing normal cognitive performance to investigate association of σ1R deficiency with AD risk. Herein we report that a single injection (i.c.v.) of Aβ25–35 impaired spatial memory with approximately 25% death of pyramidal cells in the hippocampal CA1 region of WT mice (Aβ25–35-WT mice), whereas it did not cause such impairments in σ1R+/– mice (Aβ25–35-σ1R+/– mice). Compared with WT mice, Aβ25–35-WT mice showed increased levels of NMDA-activated currents (INMDA) and NR2B phosphorylation (phospho-NR2B) in the hippocampal CA1 region at 48 h after Aβ25–35-injection (post-Aβ25–35) followed by approximately 40% decline at 72 h post-Aβ25–35 of their respective control levels, which was inhibited by the σ1R antagonist NE100. In Aβ25–35-WT mice, the administration of NR2B inhibitor Ro25-6981 or NE100 on day 1–4 post-Aβ25–35 attenuated the memory deficits and loss of pyramidal cells. By contrast, Aβ25–35-σ1R+/– mice showed a slight increase in the INMDA density and the phospho-NR2B at 48 h or 72 h post-Aβ25–35 compared to σ1R+/– mice. Treatment with σ1R agonist PRE084 in Aβ25–35-σ1R+/– mice caused the same changes in the INMDA density and the phospho-NR2B as those in Aβ25–35-WT mice. Furthermore, Aβ25–35-σ1R+/– mice treated with the NMDA receptor agonist NMDA or PRE084 on day 1–4 post-Aβ25–35 showed a loss of neuronal cells and memory impairment. These results indicate that the σ1R deficiency can reduce Aβ25–35-induced neuronal cell death and cognitive deficits through suppressing Aβ25–35-enhanced NR2B phosphorylation.