We have previously documented that enforced activation of Notch1 signaling by transient expression of the intracellular portion of the Notch transmembrane protein (NIC) induces cell growth arrest of human iliac venous and arterial endothelial cells (HIAECs). To study whether stimulation of Notch1 receptors on the cell surface results in proliferation inhibition of HIAECs, we tested two soluble forms of the Notch1 ligands, sDll4 and sJag1, for their effects on cell proliferation. We constructed sDll4 as a chimeric fusion gene, in which the cDNA encoding the extracellular domain was fused to the Fc region of the human IgG1 heavy-chain gene. Expression of the sDll4 protein was confirmed by immunoprecipitation and immunoblotting assays. The biological effects of sDll4 and sJag1 on the activation of cell surface Notch1 were validated by detecting a cleaved form of Notch1 in cells. HIAECs treated with sDll4 or sJag1 displayed reduced proliferation rates (15–18%) compared to those treated or untreated with controls. This finding implicates suppressed activation of Notch1 signaling on HIAEC proliferation. To determine whether sustained activation of the Notch1 pathway achieves a similar effect, we constructed a lentiviral vector encoding the NIC gene and transfected it into HMVECs and HIAECs. We found that proliferation rates of NIC-transfected cells markedly decreased compared with the rates of either parental or GFP-transfected control cells. These results are consistent with sDll4-initiated Notch pathway activation, but enforced constitutive activation of the Notch1 pathway has a more profound inhibitory effect on cell proliferation. Taken together, our data demonstrate that activation of the Notch1 pathway inhibits human endothelial cell proliferation in vitro.