After cutaneous injury, keratinocytes secrete paracrine factors that regulate wound cell functions; dysregulation of this signaling can lead to wound pathologies. Previously, we established that keratinocyte integrin α3β1 promotes wound angiogenesis through paracrine stimulation of endothelial cells. We hypothesize here that α3β1-dependent paracrine signaling from keratinocytes regulates the differentiation state of myofibroblasts. We report that epidermal α3-knockout mice exhibit more wound myofibroblasts and fewer cyclooxygenase 2 (Cox-2)-positive dermal cells than controls. We also found that conditioned medium from α3-expressing mouse keratinocytes (MKα3+), but not from α3-null MK cells (MKα3-), induces expression of Cox-2 in fibroblasts in a time- and dose-dependent manner and that this induction is mediated by IL-1α. Compared with MKα3- cells, MKα3+ cells secrete more IL-1α and less IL-1RA, a natural IL-1 receptor antagonist. Treatment with an IL-1α neutralizing antibody, recombinant IL-1RA, or IL-1 receptor-targeting small interfering RNA suppresses MKα3+ conditioned medium-dependent induction of Cox-2 expression in fibroblasts. Finally, active recombinant IL-1α is sufficient to induce Cox-2 in fibroblasts and to inhibit transforming growth factor-β-induced α-SMA expression. Our findings support a role for keratinocyte integrin α3β1 in controlling the secretion of IL-1α, a paracrine factor that regulates the wound myofibroblast phenotype.