A hallmark of acute respiratory distress syndrome (ARDS) is pulmonary vascular permeability. In these settings, loss of barrier integrity is mediated by cell-contact disassembly and actin-remodelling. Studies into molecular mechanisms responsible for improving microvascular barrier function are therefore vital in the development of therapeutic targets for reducing vascular permeability in ARDS. The sweet taste receptor, T1R3 is a GPCR, activated following exposure to sweet molecules, to trigger a gustducin-dependent signal cascade. In recent years, extraoral locations for T1R3 have been identified however, no studies have focused on T1R3 within the vasculature. We hypothesise that activation of T1R3, in the pulmonary vasculature, plays a role in regulating endothelial barrier function in settings of ARDS. Our study demonstrated expression of T1R3 within the pulmonary vasculature, with a drop in expression levels following exposure to barrier disruptive agents. Exposure of lung microvascular endothelial cells to the intensely sweet molecule, sucralose, attenuated LPS- and thrombin-induced endothelial barrier dysfunction. Likewise, sucralose exposure attenuated bacteria-induced lung edema formation in vivo. Inhibition of sweet taste signalling, through zinc sulfate, T1R3 or G-protein siRNA, blunted the protective effects of sucralose on the endothelium. Sucralose significantly reduced LPS-induced increased expression or phosphorylation of key signalling molecules, Src, PAK, MLC2, HSP27 and p110αPI3K. Activation of T1R3, by sucralose, protects the pulmonary endothelium from edemagenic-agent-induced barrier disruption, potentially through abrogation of Src/PAK/p110αPI3K-mediated cell-contact disassembly and Src/MLC2/HSP27-mediated actin-remodelling. Identification of sweet taste sensing in the pulmonary vasculature may represent a novel therapeutic target to protect the endothelium in settings of ARDS.