Numerous medicinal plants and their derivatives have been reported to prevent obesity and related diseases. Although Syzygium aromaticum has traditionally been used as an anodyne, carminative and anthelmintic in Asian countries, its potential in the prevention and treatment of obesity has not yet been explored. Therefore, the present study investigated the anti-obesity effect of S. aromaticum ethanol extract (SAE) both in vitro and in vivo. To evaluate the anti-obesity potential of SAE in vitro, the effect of SAE treatment on adipocyte differentiation in 3T3-L1 cells was investigated. To evaluate its potential in vivo, mice were assigned to three groups: a group fed the American Institute of Nutrition AIN-76A diet (normal group), an experimental group fed a high-fat diet (HFD group) and an experimental group fed an HFD supplemented with 0.5% (w/w) SAE (HFD + SAE group). After 9 weeks of feeding, the body weight; white adipose tissue (WAT) mass; serum triglyceride (TG), total cholesterol (TC), high-density lipoprotein (HDL) cholesterol, glucose, insulin and leptin; hepatic lipid accumulation; and levels of lipid metabolism-related genes in the liver and WAT were measured. In vitro investigation of the effect of SAE treatment on 3T3-L1 cells revealed that it had efficiently inhibited the conversion of cells into adipocytes in a dose-dependent manner. In vivo investigation revealed that SAE supplementation had significantly decreased HFD-induced increases in the body weight, liver weight, WAT mass, and serum TG, TC, lipid, glucose, insulin and leptin levels. Consistent with its effects on liver weight and WAT mass, SAE supplementation was found to have suppressed the expression of lipid metabolism-related proteins, including SREBP-1, FAS, CD36 and PPARγ in the liver and WAT, in addition to downregulating mRNA levels of transcription factors including Srebp and Pparg. SAE inhibits fat accumulation in HFD-fed mice via the suppression of transcription factors integral to adipogenesis and lipogenesis, suggesting its potential in preventing obesity.