Receptors for advanced glycation end‐products (RAGE) is transmembrane receptor of the immunoglobulin superfamily with an extracellular domain that consists of a variable region as well as two constant regions. RAGE and its ligand interaction are often referred to result in pro‐inflamatory gene activation and effect in diabetic complications, Alzheimer's disease and even some tumors. V‐domain of RAGE is determinant for binding with Amyloid‐β (Aβ). RAGE‐mediated Aβ transport of circulating Aβ across blood brain barrier (BBB) is important in pathogenesis of cerebrovascular β‐amyloidosis. In this study, we show that two β‐strands of RAGE are required for interaction with Aβ. Serial deletion analysis and site‐directed mutagenesis of RAGE V domain show that the third and eighth β‐strands are important for interaction with Aβ. Wild‐type RAGE activates the NF‐κB signaling pathway in response to Aβ peptide treatment, while a RAGE mutant defective in Aβ binding does not. Furthermore, using peptide of third and eighth β‐strand of RAGE or monoclonal antibody targeting Aβ interaction interface of RAGE V domain, interaction of RAGE and Aβ is abolished and transportation of Aβ peptide across the blood brain barrier is blocked. These results provide information crucial to the development of RAGE‐derived therapeutic reagents for Alzheimer disease.