Matrix metalloproteinase-9 (MMP-9) degrades collagen and other cellular matrix proteins. After acute ischemic stroke, increased MMP-9 levels are correlated with hemorrhage, lack of reperfusion and stroke severity. Nevertheless, definitive data that MMP-9 itself causes poor outcomes in ischemic stroke are limited. In a model of experimental ischemic stroke with reperfusion, we examined whether ischemia and recombinant tissue plasminogen activator (r-tPA) therapy affected MMP-9 expression, and we used specific inhibitors to test if MMP-9 affects brain injury and recovery. After stroke, MMP-9 expression increased significantly in the ischemic vs. non-ischemic hemisphere of the brain (p<0.001). MMP-9 expression in the ischemic, but not the non-ischemic hemisphere, was further increased by r-tPA treatment (p<0.001). To determine whether MMP-9 expression contributed to stroke outcomes after r-tPA treatment, we tested three different antibody MMP-9 inhibitors. When compared to treatment with r-tPA and saline, treatment with r-tPA and MMP-9 antibody inhibitors significantly reduced brain hemorrhage by 11.3 to 38.6-fold (p<0.01), brain swelling by 2.8 to 4.3-fold (p<0.001) and brain infarction by 2.5 to 3.9-fold (p<0.0001). Similarly, when compared to treatment with r-tPA and saline, treatment with r-tPA and an MMP-9 antibody inhibitor significantly improved neurobehavioral outcomes (p<0.001), decreased weight loss (p<0.001) and prolonged survival (p<0.01). In summary, both prolonged ischemia and r-tPA selectively enhanced MMP-9 expression in the ischemic hemisphere. When administered with r-tPA, specific MMP-9 inhibitors markedly reduced brain hemorrhage, swelling, infarction, disability and death, which suggests that blocking the deleterious effects of MMP-9 may improve outcomes after ischemic stroke.