AbstractKeratoconus (KC) a disease with established biomechanical instability of the corneal stroma, is an ideal platform to identify key proteins involved in mechanosensing. This study aims to investigate the possible role of β-catenin as mechanotransducer in KC epithelium. KC patients were graded as mild, moderate or severe using Amsler Krumeich classification. Immunoblotting and tissue immunofluorescence studies were performed on KC epithelium to analyze the expression and localization of β-catenin, E-cadherin, ZO1, α-catenin, Cyclin D1, α-actinin, RhoA, Rac123. Co-immunoprecipitation (Co-IP) of β-catenin followed by mass spectrometry of mild KC epithelium was performed to identify its interacting partners. This was further validated by using epithelial tissues grown on scaffolds of different stiffness. We observed down regulation of E-cadherin, α-catenin, ZO1 and upregulation of Cyclin D1, α-actinin and RhoA in KC corneal epithelium. β-catenin Co-IP from mild KC epithelium identified new interacting partners such as StAR-related lipid transfer protein3, Dynamin-1-like protein, Cardiotrophin-1,Musculin, Basal cell adhesion molecule and Protocadherin Fat 1.β-catenin localization was altered in KC which was validatedin vitro, using control corneal epithelium grown on different substrate stiffness. β-catenin localization is dependent upon the elastic modulus of the substrate and acts as mechanotransducer by altering its interaction and regulating the barrier function in corneal epithelium.