To elucidate the mechanism of apoptosis of liver sinusoidal endothelial cells (SECs), we examined the phosphorylation status of Bad and its upstream signaling molecules during apoptosis in culture and after ischemia-reperfusion injury. Rat SECs were isolated by the immunomagnetic method, and 2 days after culture, most SECs underwent apoptosis, which was associated with decreased tyrosine phosphorylation of cellular proteins. Addition of orthovanadate (OV), a protein tyrosine phosphatase inhibitor, sustained cellular protein phosphorylation and strongly inhibited apoptosis. Bad was dephosphorylated at Ser-112 and Ser-136 during apoptosis, but the phosphorylation status of Bad was maintained in the presence of OV. OV activated the Akt, extracellular signal-regulated protein kinase, and p38 mitogen-activated protein kinase pathways, which are involved in Bad phosphorylation. In the absence of OV, depletion of Bad by RNA interference conferred resistance to apoptosis. Hepatic injury after ischemia-reperfusion was alleviated by OV treatment, with significant inhibition of SEC apoptosis. SEC apoptosis in vivo was associated with dephosphorylation of Bad, Akt, and extracellular signal-regulated protein kinase, which was blocked by OV treatment. Our data suggest that maintenance of Bad phosphorylation is important in the prevention of SEC apoptosis and that the anti-apoptotic property of OV might have therapeutic utility.