Vascular endothelial (VE)-cadherin is localized to the endothelial borders and the adherens junctions, which are regulated by changes in mitogen-activated protein (MAP) kinases, GTPases, and intracellular calcium. We previously showed that melanoma cells induce VE-cadherin disassembly through contact with human umbilical vein endothelial cells in coculture. However, the exact mechanism by which melanoma cells signal endothelial cells to induce VE-cadherin junction disassembly is not well understood. In this study, VE-cadherin junction disassembly was further examined under fluorescence microscopy. We found that melanoma-induced VE-cadherin junction disassembly and upregulation of p38 MAP kinase in endothelial cells is regulated by both soluble factors from melanomas, particularly interleukin (IL)-8, IL-6, and IL-1beta, and through vascular cell adhesion molecule-1. Neutralizing melanoma-secreted soluble factors reduced endothelial gap formation. Endothelial cells transfected with MAP kinase kinase 6, a direct activator of p38 MAP kinase, increased VE-cadherin-mediated gap formation, facilitating melanoma transendothelial migration. In contrast, endothelial cells transfected with small-interfering RNA against p38 MAP kinase expression largely prevented melanoma transendothelial migration in Boyden chamber experiments. These findings indicate that p38 MAP kinase proteins regulate VE-cadherin junction disassembly, facilitating melanoma migration across endothelial cells.