ABSTRACT b-Klotho (b-Kl), a transmembrane protein expressed in the liver, pancreas, adipose tissues, and brain, is essential for feedback suppression of hepatic bile acid synthesis. Because bile acid is a key regulator of lipid and energy metabolism, we hypothesized potential and tissuespecific roles of b-Kl in regulating plasma lipid levels and body weight. By crossing b-kl2/2 mice with newly developed hepatocyte-specific b-kl transgenic (Tg) mice, we generated mice expressing b-kl solely in hepatocytes (b-kl2/2/Tg). Gene expression, metabolomic, and in vivo flux analyses consistently revealed that plasma level of cholesterol, which is over-excreted into feces as bile acids in b-kl2/2, is maintained in b-kl2/2 mice by enhanced de novo cholesterogenesis. No compensatory increase in lipogenesis was observed, despite markedly decreased plasma triglyceride. Along with enhanced bile acid synthesis, these lipid dysregulations in b-kl2/2 were completely reversed in b-kl2/2/Tg mice. In contrast, reduced body weight and resistance to diet-induced obesity in b-kl2/2 mice were not reversed by hepatocyte-specific restoration of b-Kl expression. We conclude that b-Kl in hepatocytes is necessary and sufficient for lipid homeostasis, whereas nonhepatic b-Kl regulates energy metabolism. We further demonstrate that in a condition with excessive cholesterol disposal, a robust compensatory mechanism maintains cholesterol levels but not triglyceride levels in mice.—Kobayashi, K., Tanaka, T., Okada, S. Morimoto, Y., Matsumura, S., Manio, MCC, Inoue, K., Kimura, K., Yagi, T., Saito, Y., Fushiki, T., Inoue, H., Matsumoto, M., Nabeshima, Y.-i. Hepatocyte b-Klotho regulates lipid homeostasis but not body weight in mice. FASEB J. 30, 000–000 (2016). www. fasebj. org