Significance Fibroblast growth factor 21 (FGF21) is an endocrine hormone that regulates glucose, lipid, and energy homeostasis. To date, little is known about the regulation of trafficking and secretion of FGF21. Here, we show that mice with a mutation in the Yipf6 gene ( Klein – Zschocher [ KLZ ]; Yipf6 ^KLZ/Y ) on a high-fat diet (HFD) have higher plasma levels of FGF21 than control mice due to the increased FGF21 secretion from their hepatocytes. Yipf6 ^KLZ/Y mice are thus resistant to HFD-induced features of the metabolic syndrome. The regulation of YIPF6 on FGF21 secretion was also conserved in nonalcoholic fatty liver disease (NAFLD) patients. YIPF6 is therefore a newly identified regulator of FGF21 secretion during development of obesity and could be a target for treatment of obesity and NAFLD.