Edema is common in preeclampsia (preE), a hypertensive disorder of pregnancy. Cardiotonic steroids (CTSs) such as marinobufagenin (MBG) are involved in the pathogenesis of preE. To assess whether CTSs are involved in the leakage of lymphatic endothelial cell (LEC), we evaluated their effect on monolayer permeability of LECs (MPLEC) in culture. A rat mesenteric LECs were treated with DMSO (vehicle), and CTSs (MBG, CINO, OUB) at concentrations of 1, 10, and 100nM. Some LECs were pretreated with 1M L-NAME (N-Nitro-L-Arginine Methyl Ester) before adding 100nM MBG or cinobufotalin (CINO). Expression of -catenin and vascular endothelial (VE)-cadherin in CTS-treated LECs was measured by immunofluorescence and MPLEC was quantified using a fluorescence plate reader. Western blot was performed to measure -catenin and VE-cadherin protein levels and myosin light chain 20 (MLC20) phosphorylation. MBG (1nM) and CINO (10nM) caused an increase (p<0.05) in the MPLEC compared to DMSO while ouabain (OUB) had no effect. Pretreatment of LECs with 1M L-NAME attenuated (p<0.05) the MPLEC. The -catenin expression in LECs was downregulated (p<0.05) by MBG and CINO. However, there was no effect on the LECs tight junctions for the CINO group. VE-cadherin expression was downregulated (p<0.05) by CINO, and MLC20 phosphorylation was upregulated (p<0.05) by MBG. We demonstrated that MBG and CINO caused an increase in the MPLEC, which were attenuated by L-NAME pretreatment. The data suggest that CTSs exert their effect via nitric-oxide-dependent signaling pathway and may be involved in vascular leak syndrome of LEC lining in preE.