Amyloid beta (Aβ) peptide 40 enhances the activation of receptor for advanced glycation end products (RAGE) in immune-inflammatory diseases. RAGE exhibits several effects in the setting of numerous cardiovascular events. We hypothesized that the Aβ40/RAGE pathway is involved in the osteoblastic differentiation of the valvular interstitial cell (VIC) phenotype, and RAGE knockout intervention could reduce the calcification of aortic valve interstitial cells (AVICs) by inhibiting the extracellular-regulated kinase1/2 (ERK1/2)/nuclear factor kappa-B (NF-κB) signaling pathway. To test this hypothesis, the activation of Aβ40/RAGE pathway in human calcific AVs was evaluated with immunohistochemical staining. Cultured calcific VIC models were used in vitro. The VICs were stimulated using Aβ40, with or without RAGE small interfering ribonucleic acid (siRNA), and ERK1/2 and NF-κB inhibitors for analysis. Our data revealed that Aβ40 induced the ERK1/2/NF-κB signaling pathway and osteoblastic differentiation of AVICs via the RAGE pathway in vitro.