The ATP-binding cassette transporter G1 (ABCG1) catalyzes export of cellular cholesterol from macrophages and hepatocytes. Here we identify an additional function of ABCG1 in the regulation of adiposity in screens of the Drosophila melanogaster and the New Zealand obese (NZO) mouse genomes. Insertion of modified transposable elements of the P-family upstream of CG17646, the Drosophila ortholog of Abcg1, generated lines of flies with increased triglyceride stores. In NZO mice, an Abcg1 variant was identified in a suggestive adiposity quantitative trait locus and was associated with higher expression of the gene in white adipose tissue. Targeted disruption of Abcg1 in mice resulted in reduced body weight gain (8.42 ± 0.6 g in Abcg1^−/−vs. 13.07 ± 1.1 g in Abcg1^+/+ mice) and adipose tissue mass gain (3.78 ± 1.3 g in Abcg1^−/−vs. 9.39 ± 1.6 g in Abcg1^+/+ mice) detected over a period of 12 wk. The reduction of adipose tissue mass in Abcg1^−/− mice was associated with markedly decreased size of the adipocytes. In contrast to their wild-type littermates, male Abcg1^−/− mice exhibited no high-fat diet-induced impairment of glucose tolerance and fatty liver. Furthermore, Abcg1^−/− mice possess decreased food intake and elevated total energy expenditure (Abcg1^−/− mice, 748.1 ± 5.4 kJ/kg metabolic body mass; Abcg1^+/+ mice, 684.3 ± 5.0 kJ/kg metabolic body mass; P = 0.011), body temperature (Abcg1^−/− mice, 37.82 ± 0.29 C; Abcg1^+/+ mice, 36.83 ± 0.24 C; P < 0.05), and locomotor activity (Abcg1^−/− mice, 3655 ± 189 counts/12 h during dark phase; Abcg1^+/+ mice, 2445 ± 235 counts/12 h during dark phase; P < 0.01). Our data indicate a previously unrecognized role of ABCG1 in the regulation of energy balance and triglyceride storage.