Increasing evidence suggests that the source of dietary protein can have an impact on weight gain and fat mass during high-fat feeding in both humans and rodents. The present study examined whether dietary bovine serum albumin (BSA) as the dominant source of protein alters energy balance and adiposity associated with high-fat feeding. C57/BL6J mice were given a diet with 10% of energy from fat and 20% of energy from casein or a diet with 45% of energy from fat and either 20% of energy from casein (HFD) or BSA (HFD+BSA) for 13 weeks. The HFD+BSA diet did not significantly alter daily energy expenditure, locomotor activity and RER, but did increase cumulative energy intake and percentage of lean mass while reducing feed efficiency and percentage of fat mass when compared with the HFD (P < 0.05). In subcutaneous adipose tissue (SAT), the HFD+BSA diet increased the mRNA levels of PPAR alpha (PPARA), carnitine palmitoyltransferase 1b (CPT1b) and uncoupling protein 3 (UCP3), but reduced the mRNA level of leptin when compared with the HFD (P<0.05). The SAT mRNA levels of PPARA, CPT1b and UCP3 were negatively correlated (P<0.05) with SAT mass, which was reduced in HFD+BSA mice compared with HFD controls (P<0.01). No differences in epididymal fat mass existed between the groups. The HFD+BSA diet normalised plasma leptin and corticosterone levels compared with the HFD (P<0.05). While differences in leptin levels were associated with the percentage of fat mass (P<0.01), changes in corticosterone concentrations were independent of the percentage of fat mass (P<0.05). The data suggest that the HFD+BSA diet influences plasma leptin levels via SAT mass reduction where mRNA levels of genes linked to beta-oxidation were increased, whereas differences in plasma corticosterone levels were not related to fat mass reduction.