Introduction: Astrocytes are known to regulate a wide variety of brain endothelial cell functions. Prior work, using a mixed species co-culture system, has shown astrocyte regulation of brain capillary endothelial expression of key hemostasis factors tissue plasminogen activator (tPA) and its inhibitor, plasminogen activator inhibitor-1 (PAI-1). The purpose of this study is to define the fibrinolytic regulatory role of human astrocytes on human brain capillary endothelial cells. Materials and methods: We used a blood–brain barrier model consisting of human astrocytes grown on transwell membrane inserts and co-cultured with human brain capillary endothelial cells. Following 48 h co-culture, we analyzed both endothelial mono-cultures and astrocyte-endothelial co-cultures for expression of tPA and PAI-1 mRNA, protein, and activity. Results and conclusions: There were significant changes for both tPA and PAI-1 mRNA:tPA mRNA levels were decreased in co-cultures (55±16% of mono-cultures, p<0.0005) and PAI-1 mRNA levels were increased 144±38%, compared to mono-cultures ( p<0.005). Co-cultures produced a 54% reduction in tPA protein (12.7±3.8 vs. 27.5±7.1 ng/ml, p<0.005) and a 24% increase in PAI-1 protein (117.5±3.2 vs. 94.9±5.9 ng/ml, p<0.0005). TGF-β neutralizing antibody attenuated the observed changes in both tPA and PAI-1. These data indicate that human astrocytes regulate human brain capillary fibrinolysis in vitro by inhibiting tPA and enhancing PAI-1 expression. This regulation is mediated, in part, by transforming growth factor-β. Our findings provide further evidence for the role of astrocytes in brain-specific hemostasis regulation.