© 2019 Cambridge University Press. All rights reserved. Recently there has been a considerable rise in the frequency of metabolic diseases, such as obesity, due to changes in lifestyle and resultant imbalances between energy intake and expenditure. Whey proteins are considered as potentially important components of a dietary solution to the obesity problem. However, the roles of individual whey proteins in energy balance remain poorly understood. This study investigated the effects of a high fat diet (HFD) containing alpha-lactalbumin (LAB), a specific whey protein, or the non-whey protein casein (CAS), on energy balance, nutrient transporters expression, and enteric microbial populations. C57BL/6J mice (n = 8) were given a HFD containing either 20% CAS or LAB as protein sources or a low-fat diet (LFD) containing CAS for 10 weeks. HFD-LAB fed mice showed a significant increase in cumulative energy intake (P=0.043), without differences in body weight, energy expenditure, locomotor activity, respiratory exchange ratio or subcutaneous and epididymal adipose tissue weight. HFD- LAB intake led to a decrease in the expression of glucose transporter glut2 in the ileum (P=0.05) and in the fatty acid transporter cd36 (P<0.001) in both ileum and jejunum. This suggests a reduction of absorption efficiency within the small intestine in the HFD-LAB group. DNA from faecal samples was used for 16S rRNA-based assessment of intestinal microbiota populations; the genera Lactobacillus, Parabacteroides and Bifidobacterium were present in significantly higher proportions in the HFD-LAB group. These data indicate a possible functional relationship between gut microbiota, intestinal nutrient transporters and energy balance, with no impact on weight gain.