Many cell types are known to modulate their behavior in response to changes in material stiffness, however little is known about how stiffness effects corneal epithelial cells. This study aims to investigate the response of a corneal epithelial cell line to polydimethylsiloxane (PDMS) substrates with a range of Young’s moduli from 10 kPa to 1500 kPa. Cellular morphology, proliferation, differentiation and mechanobiology were examined. Cells grown on PDMS adopted the typical cobblestone morphology exhibited by the corneal epithelium. Proliferative markers pERK and Ki67 were higher in cells cultured on stiffer substrates compared to those on softer substrates. Material stiffness was also found to influence the cell phenotype with cells on stiffer substrates having higher cytokeratin 3 gene expression, a mature epithelial marker, while cells on softer substrates expressed more cytokeratin 14, a basal epithelial marker. Cells grown on softer substrates also displayed higher levels of focal adhesions and intermediate filaments compared to cells on stiff substrates. This research will aid in designing novel biomaterials for the culture and transplantation corneal epithelial cells.