The phenotype of the endothelial cells (ECs) in the pseudointima of transjugular intrahepatic portasystemic shunts (TIPS) and the mechanisms of pseudointima formation after TIPS were unknown. We hypothesized that TIPS were lined by hepatic sinusoidal ECs, which stimulated the migration of smooth muscle cells (SMCs) into the pseudointima and their proliferation. Studies were done with the following specific aims: (1) isolation of ECs from TIPS pseudointima and comparison of their phenotype with human cirrhotic sinusoidal and vascular ECs derived from hepatic and portal veins as well as aorta, and (2) testing of the effects of TIPS ECs on TIPS-derived SMC migration and proliferation. ECs were isolated from eight TIPS retrieved from liver explants by immunomagnetic separation using monodispersed magnetizable polystyrene beads (Dynabeads M-450) coated with Ulex Europeus 1. EC phenotypes were examined by transmission electron microscopy, factor VIII–related antigen, CD31, CD14, and CD34 expression, uptake of acetylated LDL and secretion of type IV collagen. The effects of EC-conditioned media on SMC migration and proliferation were tested in multiwell chemotaxis chambers and by cell counting, respectively. ECs were obtained from TIPS pseudointima with >95% purity. The phenotype of TIPS-derived ECs matched that of cirrhotic sinusoidal endothelium (both expressed CD14) and differed from that of vascular endothelium (CD14 negative, Weibel-Palade positive). Conditioned media from both stenosed (n = 3) and nonstenosed (n = 3) TIPS-derived endothelial cells produced a marked (>100%) P < .001 increase in migration as well as (up to 88%) P < .01 proliferation of SMCs from both stenosed (n = 3) as well as nonstenosed TIPS (n = 3). These data indicate that TIPS pseudointima are lined by hepatic sinusoidal endothelial cells, which stimulate pseudointima formation by increasing SMC migration and proliferation.