β-Catenin in hepatocytes, under the control of Wnts, regulates pericentral gene expression. It also contributes to liver regeneration (LR) after partial hepatectomy (PH) by regulating cyclin-D1 gene expression as shown in the β-catenin and Wnt coreceptors low-density lipoprotein receptor-related protein 5/6 conditional knockouts (KO). However, conditional deletion of Wntless (Wls), required for Wnt secretion, in hepatocytes, cholangiocytes, or macrophages lacked any impact on zonation, while Wls deletion in macrophages only marginally affected LR. Here, we address the contribution of hepatic endothelial cells (ECs) in zonation and LR by characterizing EC-Wls-KO generated by interbreeding Wls-floxed and lymphatic vessel endothelial hyaluronan receptor (Lyve1)-cre mice. These mice were also used to study LR after PH. While Lyve1 expression in adult liver is limited to sinusoidal ECs only, Lyve1-cre mice bred to ROSA26-Stopflox/flox-enhanced yellow fluorescent protein (EYFP) mice showed EYFP labeling in sinusoidal and central vein ECs. EC-Wls-KO mice showed decreased liver weights; lacked glutamine synthetase, cytochrome P450 2e1, and cytochrome P450 1a2; and were resistant to acetaminophen-induced liver injury. After PH, EC-Wls-KO showed quantitative and qualitative differences in cyclin-D1 expression at 24-72 hours, which led to a lower hepatocyte proliferation at 40 hours but a rebound increase by 72 hours. ECs and macrophages isolated from regenerating livers at 12 hours showed significant up-regulation of Wnt2 and Wnt9b messenger RNA; these are the same two Wnts involved in baseline β-catenin activity in pericentral hepatocytes. Conclusion: At baseline, ECs secrete Wnt proteins essential for β-catenin activation in pericentral hepatocytes. During LR, sinusoidal and central vein ECs and secondarily macrophages secrete Wnt2, while predominantly central vein ECs and secondarily macrophages are the likely source of Wnt9b. This process spatiotemporally regulates β-catenin activation in hepatocytes to induce cell proliferation. (Hepatology Communications 2018;2:845-860).