Sphingosine-1-phosphate (S1P) is an important bioactive sphingolipid involved in angiogenesis and lymphangiogenesis, 2 important processes that influence the growth, survival, and spread of tumors. S1P acts as an extracellular mediator through binding to 5 highly specific S1P receptors, S1P1–5. Sphingosine kinase-1 (SK1), one of 2 known sphingosine kinase enzymes responsible for S1P production, appears to be overexpressed in many tumors. Although a role for S1P in angiogenesis and lymphangiogenesis has been established, it is unclear whether S1P secreted from cancer cells has a paracrine function in a tumor environment. Here we investigated whether modulation of cellular SK1 could initiate a paracrine angiogenic and lymphangiogenic switch. We found that SK1 overexpression in HEK cells or its down-regulation in glioma or breast cancer cells modulated extracellular S1P levels accordingly, which in turn increased or decreased both migration and tube formation in cocultured vascular or lymphatic endothelial cells. In contrast, down-regulation of sphingosine kinase 2 in both glioma and breast cancer cells had no appreciable effect on cellular or secreted S1P levels. In addition, vascular endothelial growth factors VEGF and VEGF-C down-regulation in cancer cells appeared insufficient to block the angiogenic and lymphangiogenic switch triggered by these cells. Moreover, S1P initiated endothelial cell sprouting in 3-dimensional collagen matrices, which is representative of a multistep angiogenic process. Our data collectively demonstrate for the first time that SK1 plays an essential role in regulating in vitro paracrine angiogenesis and lymphangiogenesis.—Anelli, V., Gault, C. R., Snider, A. J., Obeid, L. M. Role of sphingosine kinase-1 in paracrine/transcellular angiogenesis and lymphangiogenesis in vitro.