Digitalis has been used to treat congestive heart failure for more than 200 years, although the dual effects (proliferation and death) induced by digitalis on cell growth have been known for many years, the mechanisms by which digitalis causes the actions were not completely known. The aim of this work was to characterize the proliferative effect of ouabain on cell growth in endothelial cells, and, to do the differential proteomic analysis of human umbilical vein endothelial cells (HUVEC) in response to ouabain and examine changes in protein expression. HUVEC were exposed to different concentrations (0.1-100 nM) of ouabain at 12-48 h intervals. Cell growth and morphological changes of HUVEC treated with ouabain were compared with cells under nontreated conditions. Ouabain stimulated HUVEC cell proliferation at low concentrations and induced cell death at higher concentrations. Using proteomics study, we identified 32 proteins of HUVEC with various important cellular functions and revealed 8 proteins such as Annexin A1, Annexin A2, Malate dehydrogenase, Myosin regulatory light chain 2 (MRLC2), Profilin-1, S100 calcium-binding protein A13, Triosephosphate isomerase and Translationally controlled tumor protein, regulated by low-dose ouabain treatment and MRLC2 was subsequently confirmed by Western blot. Our results give new insights into the cellular and molecular mechanisms of the proliferation action of low-dose ouabain on HUVEC and provide new avenues for the treatment of cardiovascular diseases.