Objective Alzheimer's disease (AD) is a neurodegenerative disorder hallmarked by progressive memory loss and cognitive decline, linked to amyloid β-protein (Aβ) accumulation and tau hyperphosphorylation. Growing evidence implicates dysregulated N-methyl-D-aspartate receptors (NMDARs), particularly GluN2B subunit overactivation, in exacerbating excitotoxicity, synaptic loss, and neuronal death. Berberine hydrochloride (BBR), a natural isoquinoline alkaloid, demonstrates neuroprotective effects in AD models, but its precise mechanisms targeting NMDAR-mediated excitotoxicity remain underexplored. This study elucidates BBR's therapeutic potential in ameliorating AD pathology through GluN2B inhibition using in vitro and in vivo models.Methods Molecular docking simulations predicted BBR's high-affinity binding (-8.08 kcal/mol) to the GluN2B pore region, interacting with Asp616 and residues 640-652. In vitro, PC12 cells were exposed to glutamate/glycine-induced excitotoxicity, and the protective effects of BBR were assessed using CCK-8 viability assay, Fluo-3 AM Ca2? influx, JC-1 mitochondrial potential, and DCFH-DA ROS measurements. In vivo, 5×FAD mice were fed BBR orally for 3 months. Cognitive function was evaluated using the Novel Object Recognition (NOR) and Morris Water Maze (MWM) tests. Hippocampal histology (Nissl staining, Golgi-Cox dendritic analysis) assessed neuronal survival and spine density. Western blotting quantified Aβ1-42, APP, phosphorylated Tau, NMDAR subunits, PSD95, nNOS, and Caspase-1 in the hippocampal tissues.Results Molecular docking revealed a binding affinity of -8.08 kcal/mol between BBR and GluN2B, with interaction sites at Asp616 and residues 640-652. In PC12 cells, BBR significantly reversed Glu-induced viability reduction, inhibited calcium influx and ROS overproduction, and restored mitochondrial membrane potential. BBR markedly downregulated AD-related indices in vivo. Behaviorally, BBR improved spatial learning and memory in 5×FAD mice. Histological analyses showed increased Nissl body counts and dendritic spine density in the hippocampal CA1/CA3 regions. Western blotting demonstrated that BBR downregulated Aβ1-42, APP, p-Tau, and phosphorylated GluN2B, while upregulating GluN2A and PSD95 expression; it also reduced nNOS and Caspase-1 levels.Conclusion BBR effectively alleviates AD-like pathology through dual mechanisms. It inhibits GluN2B to mitigate Ca2?-driven excitotoxicity and modulation of NMDAR subunit balance and downstream signaling to promote synaptic resilience. This study establishes BBR as a promising, multi-targeted therapeutic candidate for AD, addressing both Aβ pathology and glutamatergic dysregulation. Future translational studies should explore its clinical potential in AD patients.