Transendothelial migration of malignant cells plays an essential role in tumor progression and metastasis. The present study revealed that treating human umbilical vein endothelial cells (HUVECs) with exosomes derived from metastatic breast cancer cells increased the number of cancer cells migrating through the endothelial cell layer and impaired the tube formation of HUVECs. Furthermore, the expression of intercellular junction proteins, including vascular endothelial cadherin (VE-cadherin) and zona occluden-1 (ZO-1), was reduced significantly in HUVECs treated with carcinoma-derived exosomes. Proteomic analyses revealed that thrombospondin-1 (TSP1) was highly expressed in breast cancer cell MDA-MB-231-derived exosomes. Treating HUVECs with TSP1-enriched exosomes similarly promoted the transendothelial migration of malignant cells and decreased the expression of intercellular junction proteins. TSP1-down regulation abolished the effects of exosomes on HUVECs. The migration of breast cancer cells was markedly increased in a zebrafish in vivo model injected with TSP1-overexpressing breast cancer cells. Taken together, these results suggest that carcinoma-derived exosomal TSP1 facilitated the transendothelial migration of breast cancer cells via disrupting the intercellular integrity of endothelial cells.Keywords: breast cancer; endothelial intercellular junctions; exosome; thrombospondin-1 (TSP1); transendothelial migration.