Cocaine exposure induces long-lasting molecular and structural adaptations in the brain. In this study, we show that tissue plasminogen activator (tPA), an extracellular protease involved in neuronal plasticity, modulates the biochemical and behavioral response to cocaine. When injected in the acute binge paradigm, cocaine enhanced tPA activity in the amygdala, which required activation of corticotropin-releasing factor type-1 (CRF-R1) receptors. Compared with WT mice, tPA-/- mice injected with cocaine displayed attenuated phosphorylation of ERK, cAMP response element binding protein (CREB), and dopamine and cAMP-regulated phosphoprotein 32 kDa (DARPP-32) and blunted induction of immediate early genes (IEGs) c-Fos, Egr-1, and Homer 1a in the amygdala and the nucleus accumbens (NAc). tPA-/- mice also displayed significantly higher basal preprodynorphin (ppDyn) mRNA levels in the NAc in comparison to WT mice, and cocaine decreased ppDyn mRNA levels in tPA-/- mice only. Cocaine-induced locomotor sensitization and conditioned place preference (CPP) were attenuated in tPA-/- mice. Cocaine exposure also had an anxiolytic effect in tPA-/- but not WT mice. These results identify tPA as an important and novel component of the signaling pathway that modulates cocaine-induced changes in neuroadaptation and behavior.