Pro-inflammatory processes initiated in the endothelium represent a crucial step in the pathogenesis of inflammatory cardiovascular disease, such as atherosclerosis. Recent observations pointed to potential anti-inflammatory properties of the cardiac glycoside digitoxin. Therefore, the present study investigated potential anti-inflammatory and vasoprotective properties of digitoxin as well as the underlying signaling pathways affected in endothelial cells (EC). Digitoxin, employing therapeutical concentrations used in patients (3-30nM), potently inhibited the IL-1beta-induced expression of MCP-1 and VCAM-1 in EC and the capacity of corresponding cell culture supernatants on monocyte migration as well as monocyte adhesion to endothelial monolayers, respectively. Furthermore, digitoxin prevented the IL-1beta-induced activation of p44/42-MAPK and NF-kappaB without affecting activation of JNK and p38-MAPK. Inhibition of NF-kappaB signaling but not p44/42-MAPK mimicked the observed inhibitory effects of digitoxin on MCP-1 expression and monocyte migration. Moreover, digitoxin inhibited NF-kappaB signaling at the level of TAK-1/IKK. Additionally, digitoxin prevented TNF-alpha-induced apoptosis in EC accompanied by activation of Akt. Blockade of PI-3-kinase, activator of Akt, prevented the anti-apoptotic properties of digitoxin and impaired its inhibitory action on NF-kappaB signaling and MCP-1 expression. Finally, digitoxin activated endothelial NO-synthase, which was blocked by inhibition of PI-3-kinase, Ca(2+)/Calmodulin-dependent-proteinkinase-II and chelation of intracellular calcium. Digitoxin elicits anti-inflammatory and vasoprotective properties by blocking NF-kappaB and activating PI-3-kinase/Akt signaling as well as Ca(2+)/Calmodulin-dependent-proteinkinase-II in EC. These observations indicate a potential therapeutical application of digitoxin in the treatment of cardiovascular diseases, such as atherosclerosis.