Rapamycin (Rap) has been demonstrated to affect lipid metabolism through stimulating lipolysis, inhibiting de novo lipogenesis and reducing adiposity. In the present study, we investigated rapamycin exposure’s influence on adipose tissue browning in high-fat diet-induced fatty mice. Four-week old C57BL/6J mice were fed normal chow or high-fat diet for a period of 6 weeks and then divided into three groups: (1) Nor group: mice fed with normal chow; (2) high fat diet (HFD) group: fatty mice fed with high-fat diet; (3) Rap group: high-fat diet-fed fatty mice treated intragastrically with rapamycin at a dose of 2.5 mg/kg per day for 5 weeks. Body weights and food intakes of the mice were recorded weekly. At the end of the study, blood samples were collected for glucose, lipid and insulin evaluations. Adipose tissues were weighed and lipid contents were monitored. Moreover, real-time PCR and Western blotting were applied to detect the expression levels of beige and brown fat marker genes in white adipose tissue (WAT) and brown adipose tissue (BAT). Our data demonstrated that Rap exposure significantly ameliorated metabolic defects including hyperglycaemia, dyslipidaemia and insulin resistance in the fatty mice. Furthermore, Rap treatment led to decreased tissue weights and lipid contents both in WAT and BAT. Remarkably, expression levels of BAT marker genes including uncoupling protein-1 (UCP-1), cell death-inducing DNA fragmentation factor-alpha-like effector A (CIDEA), PR-domain containing protein-16 (PRDM16) and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) were significantly down-regulated in Rap-treated fatty mice. This report demonstrates Rap exposure is capable of inhibiting adipose tissue browning in high-fat diet-induced fatty mice, and provides evidence for deeper understanding of Rap’s influence on lipid homeostasis.