ICAM-1 is required for firm adhesion of leukocytes to the endothelium. However, how the spatial organization of endothelial ICAM-1 regulates leukocyte adhesion is not well understood. In this study, we identified the calcium-effector protein annexin A2 as a novel binding partner for ICAM-1. ICAM-1 clustering promotes the ICAM-1-annexin A2 interaction and induces translocation of ICAM-1 into caveolin-1-rich membrane domains. Depletion of endothelial annexin A2 using RNA interference enhances ICAM-1 membrane mobility and prevents the translocation of ICAM-1 into caveolin-1-rich membrane domains. Surprisingly, this results in increased neutrophil adhesion and transendothelial migration under flow conditions and reduced crawling time, velocity, and lateral migration distance of neutrophils on the endothelium. In conclusion, our data show that annexin A2 limits neutrophil transendothelial migration by organizing the spatial distribution of ICAM-1.