We have previously reported that the steroid hormone estrogens stimulate activation of sphingosine kinase 1 (SphK1) and sphingosine-1-phosphate (S1P) receptors in breast cancer cells. Both estrogens and S1P are potent biological modulators of endothelial function in vasculature able to activate multiple effectors, including endothelial nitric oxide synthase (eNOS). In this study we report that treatment of endothelial cells (ECs) with 17β-estradiol (E2) resulted in a rapid, transient, and dose-dependent increase in SphK activity and increased S1P production. The effect was not reproduced by the inactive E2 analogue 17α-E2. Expression of the dominant-negative mutant SphK1G82D or transfection of SphK1-targeted siRNA in ECs caused not only a defect in SphK activation by E2, but also a significant inhibition of E2-induced activation of Akt/eNOS. Furthermore, E2 treatment induced internalization of plasma membrane S1P1 receptor, accompanied with an increase in the amount of cytosolic S1P1. By down-regulating S1P1 receptor expression, the S1P1-specific antisense oligonucleotides significantly inhibited E2-induced activation of Akt/eNOS in ECs. E2-induced EC migration and tube formation were also inhibited by S1P1 down-regulation. Thus, the findings indicate an important role of the SphK1/S1P1 pathway in mediating estrogen signaling and its actions in vasculature.