Endothelial cells are actively involved in regulating the exchanges between blood and tissues. This function is tightly dependent on actin cytoskeleton dynamics and is challenged by a wide variety of stimuli, including oxidative stress. In endothelial cells, oxidative stress quickly activates the extracellular-signal-regulated kinase (ERK) MAP kinase, which results in the phosphorylation of tropomyosin. Here, we investigated further the mechanisms of tropomyosin phosphorylation and its function in actin remodeling. We identified, for the first time, death-associated protein kinase 1 (DAP kinase 1) as the kinase that phosphorylates tropomyosin-1 in response to ERK activation by hydrogen peroxide (H2O2). We also report that the phosphorylation of tropomyosin-1 mediated by DAP kinase occurs on Ser283. Moreover, the expression of the pseudophosphorylated tropomyosin mutant Ser283Glu triggers by itself the formation of stress fibers in untreated cells, and the effect is maintained in H2O2-treated cells in which DAP kinase expression is knocked-down by siRNA. By contrast, the expression of the nonphosphorylatable tropomyosin mutant Ser283Ala is not associated with stress fibers and leads to membrane blebbing in response to H2O2. Our finding that tropomyosin-1 is phosphorylated downstream of ERK and DAP kinase and that it helps regulate the formation of stress fibers will aid understanding the role of this protein in regulating the endothelial functions associated with cytoskeletal remodeling.