We have demonstrated previously that focal adhesion kinase (FAK)/RhoA alteration by the aryl-hydrocarbon receptor (AhR) agonist 3-methylcholanthrene (3MC) is involved in the antimigratory effects of 3MC in human umbilical vascular endothelial cells. Here, we identified that signaling properties and molecular mechanisms of RhoA/beta-catenin were both implicated in alterations to blood-brain barrier integrity. The mechanisms of action were the down-regulation of integrin, the extracellular matrix, and adherens junction stability. PTEN phosphorylation by 3MC-mediated AhR/RhoA activation increased the proteasomal degradation of beta-catenin through PKCdelta/pGSK3beta-mediated beta-catenin phosphorylation; the crucial roles of AhR/RhoA in this process were verified by using gain- or loss-of-function experiments. The decrease in beta-catenin led to decreased expression of fibronectin and alpha5beta1 integrin. Additionally, protein interactions among FAK, VE-cadherin, vinculin, and beta-actin were simultaneously decreased, resulting in adherens junction instability. Novel functional TCF/LEF1 binding sites in the promoter regions of fibronectin and alpha5/beta1 integrin were identified by electrophoretic mobility shift and chromatin immunoprecipitation assays. The results indicate that the binding activities of beta-catenin decreased in mouse cerebrovascular endothelial cells treated with 3MC. In addition, simvastatin and pravastatin treatment reversed 3MC-mediated alterations in mouse cerebrovascular endothelial cells by RhoA inactivation, and the in vitro findings were substantiated by an in vivo blood-brain barrier assay. Thus, endothelial barrier dysfunction due to 3MC occurs through AhR/RhoA-mediated beta-catenin down-regulation, which is reversed by simvastatin treatment in vivo.