Objective: The aim of this study was to investigate sclerostin (SOST) expression in a rat model of experimental tympanosclerosis (TS) and its possible role in the formation of TS. Materials and Methods: Thirty-four SD rats were randomly divided into 2 groups: experimental group (n = 17) and normal group (n = 17). The left tympanic cavities in the experimental group were inoculated with methicillin-resistant Staphylococcus aureus. The changes of tympanic membranes were examined and recorded under otoendoscope. Haematoxylin-eosin staining was adopted to detect the morphological changes in the tympanic membrane and middle ear mucosa. Immunohistochemistry and Western blot analysis were used to observe the expression of SOST, Wnt3a, beta-catenin, and P-ERK1/2. Results: In the experimental group, sclerotic lesions were observed in 54.5% ears in the end of 6 weeks. Morphological changes such as mucosa incrassation, inflammatory cells infiltration, fibrous tissue proliferation, and interstitial tissue incrassation prominently appeared in the tympanic membrane and middle ear mucosa. SOST protein was mainly distributed in the cytoplasm of epithelial cells and gland cells, the expression of which increased significantly in the calcified experimental ears. In addition, expression levels of Wnt3a, beta-catenin, and P-ERK1/2 increased significantly in the calcified group too. Conclusion: The upregulated expression level of SOST may be involved in the formation of TS, first, through the pro-phosphorylation of ERK1/2 in the inflammatory stage, and then through the enhancement of Wnt3a in the osteogenic stage.