The hepatic phosphatidylcholine (PC) transporter ATP-binding cassette (ABC) B4 flops PC from hepatocytes into bile, and its dysfunction causes chronic cholestasis and fibrosis. Because a nuclear receptor-dependent PC pathway has been determined to exert antidiabetic effects, we now analyzed the role of ABCB4 in glucose metabolism. We bred congenic Abcb4-knockout (Abcb4(-/-)) mice on the fibrosis-susceptible BALB/cJ background. Knockout mice and wild-type controls were phenotyped by measuring plasma glucose concentrations, intraperitoneal glucose tolerance, hepatic RNA expression profiles, and liver histology. In addition, 4 procholestatic ABCB4 gene variants were correlated with blood glucose levels in 682 individuals from 2 independent European cohorts. Systemic glucose levels differ significantly between Abcb4(-/-) mice and wild-type controls, and knockout mice display improved glucose tolerance with significantly lower area under the curve values on intraperitoneal glucose challenge. Of note, hepatic expression of the antidiabetic nuclear receptor 5A2 (LRH-1) is induced consistently in Abcb4(-/-) mice, and its specific rare PC ligands are detected in liver by mass spectrometry imaging. In humans, serum glucose levels are associated significantly with the common ABCB4 variant c.711A>T. In summary, ABCB4 might play a critical role in glucose homeostasis in mice and humans. We speculate that the effects could be mediated via LRH-1-dependent PC pathways.