Liver sinusoidal endothelial cells (LSECs) represent a highly differentiated cell type that lines hepatic sinusoids. LSECs form a discontinuous endothelium due to fenestrations under physiological conditions, which are reduced upon chronic liver injury. Cultivation of rodent LSECs associates with a rapid onset of stress-induced senescence a few days post isolation, which limits genetic and biochemical studies ex vivo. Here we show the establishment of LSECs isolated from p19ARF-/- mice which undergo more than 50 cell doublings in the absence of senescence. Isolated p19ARF-/- LSECs display a cobblestone-like morphology and show the ability of tube formation. Analysis of DNA content revealed a stable diploid phenotype after long-term passaging without a gain of aneuploidy. Notably, p19ARF-/- LSECs express the endothelial markers CD31, vascular endothelial growth factor receptor (VEGFR)-2, VE-cadherin, von Willebrand factor, stabilin-2 and CD146 suggesting that these cells harbor and maintain an endothelial phenotype. In line, treatment with small molecule inhibitors against VEGFR-2 caused cell death, demonstrating the sustained ability of p19ARF-/- LSECs to respond to anti-angiogenic therapeutics. From these data we conclude that loss of p19ARF overcomes senescence of LSECs, allowing immortalization of cells without losing endothelial characteristics. Thus, p19ARF-/- LSECs provide a novel cellular model to study endothelial cell biology.