The wound microenvironment comprises constituents, such as the extracellular matrix (ECM), that regulate with temporal and spatial precision, the migratory, proliferative, and contractility of wound cells. Prompt closure of the wound is an early and critical phase of healing and β1 integrins are important in this process. We previously reported a marked increase in integrin α9β1 expression in epidermal keratinocytes in cutaneous and corneal wounds. However, the functional role of keratinocyte α9β1 during re-epithelialization is unknown and analysis has been precluded by the lethal phenotype of integrin α9β1 knockout mice. We now report that in conditional integrin α9 knockout (K14-α9 null) mice, normal proliferation occurs in epidermal keratinocytes and corneal basal cells. Normal epidermal keratinocyte morphology is also retained. However, corneal basal cell morphology and epithelial thickness are altered, suggesting that loss of integrin α9β1 results in abnormal corneal differentiation. In cutaneous wounds, the number of proliferating epidermal keratinocytes is significantly reduced in K14-α9 null mice compared with α9^fl/- mice, but not in Cre (control) mice. The decreased keratinocyte proliferation observed in K14-α9 null mice negatively impacts healing, resulting in a thinner migrating epithelium, demonstrating that α9β1 is crucial for efficient and proper re-epithelialization during cutaneous wound healing.