Traumatic brain injuries cause vascular hyperpermeability. Tumor necrosis factor-α (TNF-α), matrix metalloproteinase-9 (MMP-9), and caspase-3 may be important in these processes but the relationship between them has not been investigated. We hypothesized that TNF-α regulates caspase-3-mediated hyperpermeability and blood brain barrier damage and hyperpermeability directly or indirectly via activation of MMP-9. To test this, rat brain microvascular endothelial cells were treated with TNF-α with or without inhibition of MMP-9. Monolayer permeability was measured, zonula occludens-1 and F-actin configuration were examined, and MMP-9 and caspase-3 activities were quantified. TNF-α increased monolayer permeability, damaged zonula occludens-1, induced filamentous-actin stress fiber formation, and increased both MMP-9 and caspase-3 activities. Inhibition of MMP-9 attenuated these changes. These data highlight a novel link between TNF-α and MMP-9 and show that TNF-α regulated caspase-3-mediated hyperpermeability and vascular damage may be linked to MMP-9 in vitro. These findings augment the understanding of traumatic brain injury and pave the way for improved treatment.