5,6-Dimethylxanthenone-4-acetic acid (DMXAA), a potent vascular disrupting agent, selectively destroys established tumor vasculature, causing a rapid collapse in blood flow that ultimately leads to inhibition of tumor growth. Here, we demonstrate that p38 MAPK is critically involved in DMXAA-induced cytoskeleton reorganization in endothelial cells and tumor necrosis factor-α (TNF-α) production in macrophages, both of which were essential for DMXAA-induced vascular disruption. Inhibition of p38 mitogen-activated protein kinase (MAPK) significantly attenuated DMXAA-induced actin cytoskeleton reorganization in human umbilical vein endothelial cells and TNF-α production in macrophages. In vivo, p38 MAPK inhibition attenuated the immediate reduction in tumor blood flow induced by DMXAA treatment (<30 min) by inhibiting actin cytoskeleton reorganization in tumor vascular endothelial cells and blunted the long-lasting (>4 h) DMXAA-induced shutdown of the tumor vasculature by inhibiting intratumoral TNF-α production. These results indicate that p38 MAPK plays a critical role in DMXAA-induced endothelial cell cytoskeleton reorganization and TNF-α production, thus regulating DMXAA-induced antitumor activity.