Hsp27, a small heat-shock protein, has important roles in many cellular processes, including cytoskeleton dynamics, cell differentiation, and apoptosis. Its expression in normal epidermis correlates with differentiation; however, little is known about the regulatory mechanisms involved. In this study, we report that Hsp27 undergoes upregulation, phosphorylation, and redistribution to the cytoskeleton during the late phase of epidermal keratinocyte differentiation. Our results also show that the expression of the dual leucine zipper-bearing kinase (DLK), an upstream activator of the MAP kinase pathways, is sufficient by itself to induce Hsp27 phosphorylation, cell periphery localization, and redistribution to the insoluble protein fraction (cytoskeleton) in poorly differentiated keratinocytes. This redistribution correlates with the insolubilization of cornified envelope-associated proteins such as involucrin. Interestingly, the effects of DLK on Hsp27 were blocked by PD98059, a selective inhibitor of the extracellular signal-regulated protein kinase (ERK) pathway. Moreover, downregulation of Hsp27 by small interfering RNA in epithelial cells expressing DLK was accompanied by attenuated expression of involucrin in the cytoskeleton. Thus, these observations suggest that the DLK-ERK signaling pathway may act as a regulator of the interaction that occurs between Hsp27 and the cytoskeleton during the formation of the cornified cell envelope, a process conferring to the skin its crucial barrier function.