Abstract Polychlorinated biphenyl (PCB) congeners, a group of worldwide, persistent environmental contaminants, are known to cause carcinogenesis and tumor promotion, and may also affect the development of cancer metastasis. Because vascular endothelial cells create a selective barrier to the passage of cancer cells, we hypothesize that specific PCB congeners can disrupt endothelial integrity and increase the transendothelial migration of tumor cells. To examine this hypothesis, we elucidated the effects of 2,2′,4,6,6′-pentachlorobiphenyl (PCB 104), a representative of highly ortho-substituted non-coplanar PCB congeners, on the endothelial permeability and transendothelial migration of MDA-MB-231 breast cancer cells. Exposure of human microvascular endothelial cell 1 (HMEC-1) to PCB 104 induced endothelial hyperpermeability and markedly increased transendothelial migration of MDA-MB-231 cells. These effects were associated with overexpression of vascular endothelial growth factor (VEGF). PCB 104-mediated elevation of VEGF expression was induced by phosphatidylinositol 3-kinase (PI3K) but not affected by co-treatments with antioxidants or the NF-κB inhibitor SN50. In addition, the PI3K-dependent pathway was involved in PCB 104-induced activation of AP-1, a transcription factor implicated in the regulation of VEGF gene expression. The VEGF receptor (KDR/Flk-1) antagonist SU1498 and the PI3K inhibitor LY294002 inhibited PCB 104-induced hyperpermeability. These results indicate that PCB 104 may contribute to tumor metastasis by inducing VEGF overexpression that stimulates endothelial hyperpermeability and transendothelial migration of cancer cells.