Besides its role as a thrombolytic agent, tissue plasminogen activator (tPA) triggers harmful effects in the brain parenchyma after stroke, such as inflammation, excitotoxicity and basal lamina degradation. Neuroserpin, a natural inhibitor of tPA, has shown neuroprotective effects in animal models of brain infarct. However, the molecular mechanisms of neuroserpin-mediated neuroprotection after brain ischemia remain to be well characterized. Then, our aim was to investigate such mechanisms in primary mixed cortical cell cultures after oxygen and glucose deprivation (OGD). Primary rat mixed cortical cultures containing both astrocytes and neurons were subjected to OGD for 150min and subsequently treated with either tPA (5μg/mL), neuroserpin (0.125, 0.25, 0.5 or 1μM), and tPA together with neuroserpin at the mentioned doses. Twenty-four hours after treatment, LDH release, caspase-3 activity, MCP-1, MIP-2, active MMP-9, GRO/KC and COX-2 were measured. Statistical differences were analyzed using Student's t-test or one-way ANOVA as appropriate. Treatment with tPA after OGD increased LDH release, active MMP-9, MCP-1 and MIP-2 (all p≤0.05), but not caspase-3, GRO/KC or COX-2 compared to control. Treatment with neuroserpin after OGD decreased LDH release and active MMP-9 (all p≤0.05). It had no effect on caspase-3 activity, or on MCP-1, MIP-2, GRO/KC or COX-2 expression compared to control. Administration of tPA together with neuroserpin decreased LDH release, active MMP-9 and MIP-2 (all p≤0.05) and showed no effect on MCP-1, GRO/KC or COX-2 compared to control. Our results suggest that neuroprotective activity of neuroserpin involves attenuation on tPA-mediated mechanisms of inflammation and BBB disruption after brain ischemia.