The endothelial barrier function regulated by the cytoskeletal reorganizations has been implicated in the pathogenesis of multiple lung diseases including asthma, sepsis, edema, and acute respiratory distress syndrome. The extensive studies have established that activation of small GTPase Rac is a key mechanism in endothelial barrier protection but the role of microtubules-associated Rac in the endothelial functions remains poorly understood. With the emerging evidences that microtubules disassembly also plays a critical role in actin cytoskeleton remodeling leading to endothelial permeability, the knowledge on microtubules-mediated regulation of endothelial barrier is imperative to better understand the etiology of lung injuries as well as to develop novel therapeutics against these disorders. In this regard, our recent studies have revealed some novel aspects of microtubules-mediated regulation of endothelial barrier functions and unraveled a putative role of Rac-specific guanine nucleotide exchange factor Asef in mediating the barrier protective effects of hepatocyte growth factor. In this review, we will discuss the role of this novel Rac activator Asef in endothelial barrier protection and its regulation by microtubules.