<p>Alterations of fatty acyl composition in a triacylglycerol (TAG) are associated with metabolic diseases such as obesity and diabetes. This study proposes that the arrangement of fatty acyl groups in a TAG affects body weight gain and lipid metabolism in mice fed a high-fat diet (36% w/w). In the experiment, six-week-old male C57BL/6&#xa0;J mice were randomly assigned to three dietary groups: 1,2-dipalmitoyl-3-oleoylglycerol (PPO), 1,3-dipalmitoyl-2-oleoylglycerol (POP), and a regular chow control group. Food intake and body weight were monitored daily for over six weeks. The mice in the PPO group had significantly higher food and caloric intakes, more body weight gain, and lower liver-to-body weight ratio than that in the POP group. Although mice in the chow group consumed more food by weight, their caloric intake and body weight gain were lower than that in the PPO group, but similar to that in the POP group. Blood glucose levels in the PPO group were higher than that in the POP and Chow groups. Furthermore, mice in the chow group had lower cholesterol levels than those in the PPO and POP groups, with no significant differences in blood TAG levels among the three groups. The chow group had higher levels of hepatic insulin receptor substrate 1, fatty acid synthase, and phosphorylated acetyl-CoA carboxylase than the PPO and POP groups. In comparison, the POP group had lower levels of stearoyl-CoA desaturase 1 than the chow and PPO groups. Our results indicate that the arrangement of fatty acyl groups in TAG significantly influences food intake, body weight gain, and lipid homeostasis in mice.</p>