Sphingosine-1-phosphate (S1P) receptor subtype 1 (S1P(1)), a G-protein coupled receptor (GPCR), regulates many biological activities of endothelial cells (ECs). In this report, we show that S1P(1) receptors are present in the nuclei of ECs by using various biochemical and microscopic techniques such as cellular fractionation, immunogold labeling, and confocal microscopic analysis. Live cell imaging showed that plasma membrane S1P(1) receptors are rapidly internalized and subsequently translocated to nuclear compartment upon S1P stimulation. Utilizing membrane biotinylation technique further supports the notion that nuclear S1P(1) receptors were internalized from plasma membrane S1P(1) after ligand treatment. Moreover, nuclear S1P(1) is able to regulate the transcription of Cyr61 and CTGF, two growth factors functionally important in the regulation of vasculature. Collectively, these data suggest a novel S1P-S1P(1) signaling axis present in the nuclear compartment of endothelial cells, which may regulate biological responses of endothelium.