The cornea is an excellent model tissue to study how cells adapt to periods of hypoxia as it is naturally exposed to diurnal fluxes in oxygen. It is avascular, transparent, and highly innervated. In certain pathologies, such as diabetes, limbal stem cell deficiency, or trauma, the cornea may be exposed to hypoxia for variable lengths of time. Due to its avascularity, the cornea requires atmospheric oxygen, and a reduction in oxygen availability can impair its physiology and function. We hypothesize that hypoxia alters membrane stiffness and the deposition of matrix proteins, leading to changes in cell migration, focal adhesion formation, and wound repair. Two systems-a 3D corneal organ culture model and polyacrylamide substrates of varying stiffness-were used to examine the response of corneal epithelium to normoxic and hypoxic environments. Exposure to hypoxia alters the deposition of the matrix proteins such as laminin and Type IV collagen. In addition, previous studies had shown a change in fibronectin after injury. Studies performed on matrix-coated acrylamide substrates ranging from 0.2 to 50 kPa revealed stiffness-dependent changes in cell morphology. The localization, number, and length of paxillin pY118-and vinculin pY1065-containing focal adhesions were different in wounded corneas and in human corneal epithelial cells incubated in hypoxic environments. Overall, these results demonstrate that low-oxygenated environments modify the composition of the extracellular matrix, basal lamina stiffness, and focal adhesion dynamics, leading to alterations in the function of the cornea.