The present study examined the underlying mechanisms by which whey protein isolate (WPI) affects energy balance. C57BL/6J mice were fed a diet containing 10 % energy from fat, 70 % energy from carbohydrate (35 % energy from sucrose) and 20 % energy from casein or WPI for 15 weeks. Mice fed with WPI had reduced weight gain, cumulative energy intake and dark-phase V O2 compared with casein-fed mice (P,0·05); however, WPI intake had no significant effects on body composition, meal size/number, water intake or RER. Plasma levels of insulin, TAG, leptin, glucose and glucagon-like peptide 1 remained unchanged. Notably, the intake of WPI reduced stomach weight and both length and weight of the small intestine (P,0·05). WPI intake reduced the gastric expression of Wingless/int-1 5a (Wnt5a) (P, 0·01) and frizzled 4 (Fzd4) (P, 0·01), with no change in the expression of receptor tyrosine kinase-like orphan receptor 2 (Ror2) and LDL receptor-related protein 5 (Lrp5). In the ileum, WPI increased the mRNA expression of Wnt5a (P,0·01) and caused a trend towards an increase in the expression of Fzd4 (P¼0·094), with no change in the expression of Ror2 and Lrp5. These genes were unresponsive in the duodenum. Among the nutrient-responsive genes, WPI specifically reduced ileal mRNA expression of peptide YY (P,0·01) and fatty acid transporter protein 4 (P,0·05), and decreased duodenal mRNA expression of the insulin receptor (P¼ 0·05), with a trend towards a decreased expression of Na -glucose co-transporter 1 (P¼0·07). The effects of WPI on gastrointestinal Wnt signalling may explain how this protein affects gastrointestinal structure and function and, in turn, energy intake and balance.Key words: Whey proteins: Stomach: Intestine: Energy intake Whey proteins are a by-product of cheese manufacturing. In recent years, these dietary proteins have gained considerable interest in relation to their health benefits, including reduction in energy intake and body weight, and improvement in insulin sensitivity (1,2) . However, more research is required to assess whether the effects of whey protein are dependent on macronutrient composition in the diet, as demonstrated previously for diets enriched with whey protein (3) , as this will allow the formulation of diets with the optimum macronutrient combination that confers specific beneficial effects on human health. Furthermore, such analysis may also reveal the mechanisms underpinning the effects of these proteins. We have previously shown that intake of 20 % energy from whey protein isolate (WPI) reduces weight gain and fat mass, and increases lean mass in mice fed a high-fat (45 % energy) diet (4,5) . In contrast, intake of a high whey protein content (above the 14 -25 % adequate energy range) has been shown to influence energy balance in mice fed a low-fat diet (10 % energy from fat). Moreover, where an effect is found, the underlying mechanisms have not been elucidated. For instance, mice fed a diet containing 30 % energy from whey protein and 10 % energy from fat exhibited reduced...