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The current standard of care for moderate to severe ischemic stroke is thrombolytic therapy with tissue plasminogen activator (tPA). Treatment with tPA can significantly improve neurological outcomes; however, thrombolytic therapy is associated with an increased risk of intracerebral hemorrhage (ICH). The risk of hemorrhage significantly limits the use of thrombolytic therapy and identifying pathways induced by tPA that increase this risk could provide new therapeutic options to extend thrombolytic therapy to a wider patient population. Here, we investigate the role of protein kinase (PK)Cβ phosphorylation of the tight junction protein occludin during ischemic stroke and its role in cerebrovascular permeability. We demonstrate that activation of this pathway by tPA is associated with an increased risk of ICH. Middle cerebral artery occlusion (MCAO) increased occludin serine 490 (S490) phosphorylation in the ischemic penumbra in a tPA-dependent manner, as tPA-/-mice were significantly protected from MCAO-induced occludin phosphorylation. Intra-ventricular injection of tPA in the absence of ischemia was sufficient to induce occludin phosphorylation and vascular permeability in a PKCβ-dependent manner. Blocking occludin phosphorylation either by targeted expression of a non-phosphorylatable form of occludin (S490A) or by pharmacologic inhibition of PKCβ, reduced MCAO-induced permeability and improved functional outcome. Further, inhibiting PKCβ after MCAO prevented ICH associated with delayed thrombolysis. These results reveal that PKCβ phosphorylation of occludin is a downstream mediator of tPA-induced cerebrovascular permeability and suggest that PKCβ inhibitors could improve stroke outcome and prevent ICH associated with delayed thrombolysis, potentially extending the window for thrombolytic therapy in stroke.