We show in rat lung microvessel endothelial cells (RLMVEC) that endocytosis is a critical determinant of activation of p38 mitogen-activated protein kinase (p38 MAPK), and thereby regulates endothelial monolayer integrity. In RLMVEC grown in serum-free medium, we observed that albumin supplementation induced the phosphorylation of p38 MAPK within 30 min, which persisted for up to 2 hr. Engagement of the endocytic machinery regulated the activation of p38 MAPK that contributed to endothelial cell proliferation and reduction of apoptosis. We also observed an interaction between the caveolar protein, caveolin-1, and p38 MAPK with reciprocal co-immunoprecipitation assays and co-localization using double label immunofluorescence staining. Knockdown of caveolin-1 expression with siRNA significantly reduced endocytosis and activation of p38 MAPK and interfered with the ability of endothelial cells to form a confluent monolayer. Thus, caveolae-mediated endocytosis and concomitant activation of p38 MAPK may help to maintain endothelial monolayer integrity by signaling proliferation and survival of endothelial cells.