Low-carbohydrate, high-fat diets under eucaloric conditions are associated with several health-beneficial metabolic effects in humans, particularly in the liver. We recently observed that apolipoprotein A-IV (apoA-IV), a highly abundant apolipoprotein, was among the most upregulated proteins in the circulation after six weeks of high fat intake in humans. However, the dietary regulation of apoA-IV and the potential effects of apoA-IV on regulation of glucose- and lipid metabolism remain to be fully established. We here demonstrate in healthy human individuals that both short- and long-term high-fat intake increased fasting plasma apoA-IV concentrations by up to 54%, while high carbohydrate intake suppressed plasma apoA-IV concentrations. In mice, administration of apoA-IV acutely lowered blood glucose levels in lean and obese mice. Interestingly, this was related to a dual mechanism, involving both inhibition of hepatic glucose production and increased glucose uptake into white and brown adipose tissues. In addition to an effect on hepatic glucose production, the apoA-IV-induced liver proteome revealed increased capacity for lipoprotein clearance. The effects of apoA-IV in the liver and adipose tissues were obtained concomitant with increased whole-body fatty acid oxidation. Upon glucose stimulation, an improvement in glucose tolerance by apoA-IV administration was related to potentiation of glucose-induced insulin secretion, while apoA-IV inhibited glucagon secretion ex vivo in islets. In conclusion, we find that ApoA-IV is potently increased by the intake of fat in humans, and that several beneficial metabolic effects, previously associated with high fat intake in humans, are mimicked by administration of apoA-IV protein to mice.