Changes in endothelial permeability are crucial in the pathogenesis of many diseases. Adenosine is one of the endogenous mediators controlling endothelial permeability under normal conditions, and an endothelial cell surface enzyme CD73 is a key regulator of adenosine production. Here we report that IFN-beta is a novel inducer of CD73. We found that pretreatment with IFN-beta dramatically improved the vascular barrier function in lungs after intestinal ischemia-reperfusion injury in wild-type animals in vivo. IFN-beta had absolutely no protective effects in CD73-deficient mice, which suffered from more severe lung damage than wild-type mice, showing that IFN-beta functions strictly in a CD73-dependent manner. Most importantly, IFN-beta treatment initiated after the ischemic period almost completely inhibited vascular leakage during the reperfusion. IFN-beta also induced the expression and activity of CD73 and concurrently decreased vascular permeability in cultured human pulmonary endothelial cells. These data show that induction of CD73 and improvement of vascular barrier are new mechanisms for the anti-inflammatory action of IFN-beta. Moreover, IFN-beta treatment may be useful in alleviating vascular leakage induced by ischemia-reperfusion injury.