The dietary R-3-hydroxybutyrate-R-1,3-butanediol monoester increases resting energy expenditure (REE) and markers of brown and white adipose thermogenesis in lean mice. The purpose of this investigation was to determine whether the ketone ester, R,S-1,3-butanediol diacetoacetate (BD-AcAc(2)), increases energy expenditure and markers of adipose tissue thermogenesis in the context of high-fat diet (HFD)–induced obesity. Thirty-five-week–old male C57BL/6J mice were placed on an ad libitum HFD (45% kcal) for 10 wk. The mice were then randomized to 1 of 3 groups (n = 10 per group) for an additional 12 wk: 1) control (Con), continuous HFD, 2) pair-fed (PF) to ketone ester (KE); and 3) KE: HFD+30% energy from BD-AcAc(2). Mean energy intake throughout the study was ∼26% lower in the KE compared to the Con group (8.2 ± 0.5 vs. 11.2 ± 0.7 kcal/d; P < 0.05). Final body weight (26.8 ± 3.6 vs. 34.9 ± 4.8 g; P < 0.001) and fat mass (5.2 ± 1.2 vs. 11.3 ± 4.5 g; P < 0.001) of the KE group was significantly lower than PF, despite being matched for energy provisions. Differences in body weight and adiposity were accompanied by higher REE and total energy expenditure in the KE group compared to PF after adjustment for lean body mass and fat-mass (P = 0.001 and 0.007, respectively). Coupled or uncoupled mitochondrial respiratory rates in skeletal muscle were not different among groups, but markers of mitochondrial uncoupling and thermogenesis (uncoupling protein-1, deiodinase-2, and peroxisome proliferator-activated receptor γ coactivator-1α) were higher in interscapular brown adipose tissue (BAT) of mice receiving the KE diet. The absence of mitochondrial uncoupling in skeletal muscle and increased markers of mitochondrial uncoupling in BAT suggest that BD-AcAc(2) initiates a transcriptional signature consistent with BAT thermogenesis in the context of HFD-induced obesity.—Davis, R. A. H., Deemer, S. E., Bergeron, J. M., Little, J. T., Warren, J. L., Fisher, G., Smith, D. L., Jr., Fontaine, K. R., Dickinson, S. L., Allison, D. B., Plaisance, E. P. Dietary R,S-1,3-butanediol diacetoacetate reduces body weight and adiposity in obese mice fed a high-fat diet.