Objective ·To investigate the effects of intermittent fasting (IF) combined with thermogenic fat activation on the treatment and prevention of obesity in mice. Methods ·Male C57BL/6J mice aged 8 weeks were fed by high-fat diet for 4 months and then used as obesity treatment models. The prevention model was conducted on male and 8-week-old C57BL/6J mice. High-fat diet-induced obese mice and normal mice were respectively assigned into four groups: control group, alternate-day intraperitoneal CL316243 (β3-adrenergic receptor agonist,CL) injection group, IF group, and IF combined with alternate-day intraperitoneal CL injection group. Obesity treatment experimental mice and obesity prevention experimental mice were intervened for 38 d and 124 d, respectively, and they were all fed with high-fat diet during the intervention. The food intake and body weight were measured every two days. The blood glucose was measured at the end of the experiments. The brown adipose tissues (BAT), inguinal white adipose tissues (iWAT), epididymal white adipose tissues (eWAT), and livers were collected and weighed after the mice were sacrificed. The effect of IF combined with CL on morphologic changes was investigated by hematoxylin-eosin (H-E) staining. The expression levels of the genes related to thermogenesis, inflammation, and glucose and lipid metabolisms in the livers and adipose tissues were detected by real-time quantitative polymerase chain reaction (RT-qPCR). Results ·In the treatment model, compared with IF alone, IF combined with CL further reduced the body weight and blood glucose of obese mice (P<0.05), reduced the lipid droplet size in the eWAT cells and the liver cells (P<0.05), promoted the expression levels of the thermogenic genes uncoupling protein 1 (Ucp1) and cell death inducing DFFA like effector α (Cidea) in the eWAT and the iWAT, and up-regulated the expressions of the fatty acid oxidation related genes in the eWAT and the iWAT, i.e., peroxisome proliferator-activated receptor α (Ppara) and enoyl-CoA hydratase and 3-hydroxyacyl CoA dehydrogenase (Ehhadh) (P<0.05). IF combined with CL also inhibited the expressions of inflammation-related genes in the eWAT and the liver (P<0.05) and promoted the expressions of glucose metabolism-related genes in the liver compared with the control group (P<0.05), but there were no significant differences compared with IF alone. In the prevention model, compared with IF alone, IF combined with CL further reduced the lipid droplet size in the eWAT cells and the iWAT cells, promoted the expression levels of Ucp1 and Cidea in the eWAT and the iWAT, and up-regulated the expression of Ppara and Ehhadh in the eWAT and the iWAT (P<0.05). IF combined with CL also resisted the weight gain induced by high-fat diet, improved blood glucose (P<0.05), and inhibited the expression levels of fatty acid oxidation-related genes in the liver compared with the control group (P<0.05), but there were no significant differences compared with IF alone. Conclusion ·Both in the obesity treatment and prevention models, IF combined with thermogenic fat activation can reduce lipidosis in the adipose tissue and promote the expression of thermogenic genes and fatty acid oxidation genes in the white adipose tissue compared with IF alone; however, the combined effects of them on body weight and blood glucose are superior to IF in the obesity treatment model, but not in the prevention model.