In this study, we report a phospholipase C (PLC)-mediated mechanism for the redistribution of interendothelial adherens junctions in response to melanoma cell contacts with the endothelium. We demonstrated that contact of melanoma cells to human umbilical vein endothelial cells (HUVEC) triggered rapid endothelial [Ca2+]i response through PLC-IP3 pathway. In addition, alternation of endothelial adherens junctions following contact of melanoma cells was evidenced by the changes in immunological staining patterns of vascular endothelial (VE)-cadherin. A PLC inhibitor, U73122 was shown to significantly diminish [Ca2+]i response and reduce the occurrence of melanoma cell-induced VE-cadherin reorganization. Moreover, inhibition of PLC attenuated melanoma cell transendothelial migration. However, melanoma cell-associated VE-cadherin breakdown was not sensitive to Ly294002, an inhibitor of phosphatidylinositol-3-kinase (PI3K), whereas inhibition of PI3K resulted in a reduction of melanoma cell transmigration. Taken together, our findings implicate that by inducing the PLC-Ca2+ signaling pathway, melanoma cells disrupt EC junctions to breach the endothelium and promote transvascular homing of tumor cells.