Tympanosclerosis (TS) has become a common pathological condition of the middle ear, but the underlying mechanism of it is still ambiguous. It was found that osteoprotegerin/receptor activator of nuclear factor kappa B ligand (OPG/RANKL) axis played an important role in the development of TS and was regulated by the extracellular signal regulated kinase 1/2-mitogen activated protein kinase (ERK1/2-MAPK) pathway. However, whether ERK1/2-MAPK pathway mediates the occurrence of TS by regulating OPG/RANKL axis has not been reported. In this study, MAPK and calcium pathway were found significantly activated in TS model. In vivo, the expression of p-ERK1/2 in TS model was significantly increased. In vitro, osteoblasts were isolated from auditory vesicles of neonatal rats for the first time, and then cultured with ERK1/2-MAPK pathway inhibitor PD98059. As results, PD98059 showed inhibitory effects on the phosphorylation of ERK1/2 and proliferation of osteoblasts. Besides, different concentrations of PD98059 showed different inhibitory effects on mRNA expression of osteocalcin (Ocn), bone sialoprotein (Bsp), runt-related transcription factor 2 (Runx2), bone morphogenetic protein 2 (Bmp2) and OPG. Therefore, it was speculated that the ERK1/2-MAPK pathway may affect the formation of TS by regulating the proliferation and differentiation of osteoblasts, which may be helpful for the study of drug target for tympanosclerosis.Copyright © 2025. Published by Elsevier Inc.