Vascular leakage is a hallmark of the inflammatory response. Acute changes in endothelial permeability are due to posttranslational changes in intercellular adhesion and cytoskeleton proteins. However, little is known about the mechanisms leading to long-term changes in vascular permeability. Here, we show that interleukin-6 (IL-6) promotes an increase in endothelial monolayer permeability that lasts over 24 h and demonstrate that activation of Src and MEK/ERK pathways is required only for short-term increases in permeability, being dispensable after 2 h. In contrast, Janus kinase (JAK)-mediated STAT3 phosphorylation at Y705 (but not S727) and de novo synthesis of RNA and proteins are required for the sustained permeability increases. Loss of junctional localization of VE-cadherin and ZO-1 is evident several hours after the maximal IL-6 response, thus suggesting that these events are a consequence of IL-6 signaling, but not a cause of the increased permeability. Understanding the mechanisms involved in sustaining vascular permeability may prove crucial to allow us to directly target vascular leakage and minimize tissue damage, thus reducing the rates of mortality and chronic sequelae of excessive edema. Targeting endothelial-specific mechanisms regulating barrier function could provide a new therapeutic strategy to prevent vascular leakage while maintaining the immune response and other beneficial aspects of the inflammatory response that are required for bacterial clearance and tissue repair.