The blood-brain barrier (BBB) prevents the entrance of circulating molecules and immune cells into the central nervous system. The barrier is formed by specialized brain endothelial cells that are interconnected by tight junctions (TJ). A defective function of the BBB has been described for a variety of neuroinflammatory diseases, indicating that proper regulation is essential for maintaining brain homeostasis. Under pathological conditions, reactive oxygen species (ROS) significantly contribute to BBB dysfunction and inflammation in the brain by enhancing cellular migration. However, a detailed study about the molecular mechanism by which ROS alter BBB integrity has been lacking. Here we demonstrate that ROS alter BBB integrity, which is paralleled by cytoskeleton rearrangements and redistribution and disappearance of TJ proteins claudin-5 and occludin. Specific signaling pathways, including RhoA and PI3 kinase, mediated observed processes and specific inhibitors of these pathways prevented ROS-induced monocyte migration across an in vitro model of the BBB. Interestingly, these processes were also mediated by protein kinase B (PKB/Akt), a previously unknown player in cytoskeleton and TJ dynamics that acted downstream of RhoA and PI3 kinase. Our study reveals new insights into molecular mechanisms underlying BBB regulation and provides novel opportunities for the treatment of neuroinflammatory diseases.--Schreibelt, G., Kooij. G., Reijerkerk, A., van Doorn, R., Gringhuis, S. I., van der Pol, S., Weksler, B. B., Romero, I. A., Couraud, P.-O., Piontek, J., Blasig, I. E., Dijkstra, C. D., Ronken, E., de Vries, H. E. Reactive oxygen species alter brain endothelial tight junction dynamics via RhoA, PI3 kinase and PKB signaling.