Previously, we hypothesized that the biomechanical properties of the surface supporting the corneal epithelium plays a fundamental role on the tissue's homeostasis (i.e., the balance between epithelial cell migration, differentiation, proliferation, and desquamation). Here we analyse these biomechanical properties from fresh human corneas at an unprecedented resolution using Brillouin spectro‐microscopy. This non‐contact technique revealed that the different corneal tissues can be clearly distinguished, with significantly different Brillouin frequency shifts forming distinctive bulk modulus patterns between the central cornea and the limbus. Specifically, the highest modulus was observed in the sub‐epithelium matrix of the central cornea (i.e., Bowman's layer), whereas the area with lowest bulk modulus corresponded to the sub‐epithelial matrix surrounding the populations of limbal epithelial stem cells (LESCs). We then showed that, in vitro, high‐density collagen gels softened by the localized application of collagenase are capable of persistently supporting slow‐moving, proliferative CK15+/CK3− corneal epithelial cells. In contrast, untreated gel surfaces elicited quicker differentiation of LESCs. Moreover, the same technique applied in situ allowed the softening of the anterior surface of the central cornea, which was then capable of supporting the re‐epithelialisation of ABCG2+/CK15+/α9‐integrin+/CK3‐ corneal epithelial cells capable of reforming a limbus‐characteristic basement membrane (BM) containing laminin‐γ3. Together, these results support the notion that the phenotype of corneal epithelial cells depends on the mechanical properties of their residence. Following on from this we undertook a series of animal studies to test our hypothesis that the loss of limbal stem cells following alkali burn was attributable to a pathological stiffening of the limbal sub‐epithelial tissue and that a restoration of this stiffness via the pharmacological action of collagenase could reverse the loss of limbal stem cells.