Reepithelialization is a critical step in wound healing. It is initiated by keratinocyte migration at the wound edges. After wounding, extracellular nucleotides are released by keratinocytes and other skin cells. Here, we report that activation of P2Y₂ nucleotide receptor by ATP/UTP inhibits keratinocyte cell spreading and induces lamellipodium withdrawal. Kymography analysis demonstrates that these effects correlate with a durable decrease of lamellipodium dynamics. P2Y₂ receptor activation also induces a dramatic dismantling of the actin network, the loss of 3 integrin expression at the cell periphery, and the dissolution of focal contacts as indicated by the alteration of v integrins and focal contact protein distribution. In addition, activation of P2Y₂R prevents growth factor-induced phosphorylation of Erk_1,2 and Akt/PkB. The use of a specific pharmacological inhibitor (YM-254890), the depletion of G_(q/11) by siRNA, or the expression of a constitutively active G_(q/11) mutant (Q209L) show that activation of G_(q/11) is responsible for these ATP/UTP-induced effects. Finally, we report that ATP delays growth factor-induced wound healing of keratinocyte monolayers. Collectively, these findings provide evidence for a unique and important role for extracellular nucleotides as efficient autocrine/paracrine regulators of keratinocyte shape and migration during wound healing.--Taboubi, S., Milanini, J., Delamarre, E., Parat, F., Garrouste, F., Pommier, G., Takasaki, J., Hubaud, J-C., Kovacic, H., Lehmann, M. G(q/11)-coupled P2Y₂ Nucleotide Receptor Inhibits Human Keratinocyte Spreading and Migration.