Many tumor cells are capable of migrating through endothelial cell (EC) junctions and disintegrating sub-endothelial extracellular matrix to achieve extravasation. We demonstrate in this study that certain solid tumor cells can induce EC apoptosis to facilitate their escape from the circulation. The EC apoptosis is triggered by elevated intracellular reactive oxygen species (ROS) levels and direct contacts with tumor cells are required. Treating ECs with antioxidants, such as ascorbate and N-acetyl-L-cysteine (NAC), and a glutathione precursor can rescue the ECs from tumor-induced apoptosis and reduce the number of tumor cells migrating across endothelial barriers. NAD(P)H oxidase was identified as the major ROS producer in the event since inhibitors and small interference RNA specific to the enzyme could abrogate the tumor-induced ROS production and hence EC death. This study also provides evidence showing that the interaction between tumor and EC increases intracellular Ca2+ concentration and activates protein kinase C (PKC) activity, which leads to NAD(P)H oxidase activation through the serine-phosphorylation of p47phox subunit. These findings suggest that blocking the tumor-induced EC apoptosis is a potential way to prevent tumor metastasis. J. Cell. Physiol. 226: 1750–1762, 2011. © 2010 Wiley-Liss, Inc.