Cells heal disruptions in their plasma membrane using a sophisticated, efficient, and conserved response involving the formation of a membrane plug and assembly of an actomyosin ring. Here we describe how Rho family GTPases modulate the cytoskeleton machinery during single cell wound repair in the genetically amenable Drosophila embryo model.We find that Rho, Rac, and Cdc42 rapidly accumulate around the wound and segregate into dynamic, partially overlapping zones. Genetic and pharmacological assays show that each GTPase makes specific contributions to the repair process. Rho1 is necessary for myosin II activation, leading to its association with actin. Rho1, along with Cdc42, is necessary for actin filament formation and subsequent actomyosin ring stabilization. Rac is necessary for actin mobilization toward the wound. These GTPase contributions are subject to crosstalk among the GTPases themselves and with the cytoskeleton. We find Rho1 GTPase uses several downstream effectors, including Diaphanous, Rok, and Pkn, simultaneously to mediate its functions.Our results reveal that the three Rho GTPases are necessary to control and coordinate actin and myosin dynamics during single-cell wound repair in the Drosophila embryo. Wounding triggers the formation of arrays of Rho GTPases that act as signaling centers that modulate the cytoskeleton. In turn, coordinated crosstalk among the Rho GTPases themselves, as well as with the cytoskeleton, is required for assembly/disassembly and translocation of the actomyosin ring. The cell wound repair response is an example of how specific pathways can be activated locally in response to the cell's needs.