Obesity is a chronic, relapsing, complex disease characterized by an energy imbalance in adipose tissue, as well as chronic inflammation. Obesity can result in metabolic disease and is an increasing global concern. Current synthetic anti-obesity medications have been linked to gastrointestinal problems and a variety of side effects; therefore, it is essential to identify natural compounds that are effective, safe, and nontoxic for weight loss. It has been demonstrated that the brown alga Sargassum thunbergii (S. thunbergii) found in the shallow marine coasts of northwest Pacific has a number of biological functions, including an antiobesity effect. However, the role of S. thunbergii in the browning process and the composition of the gut microbiota remains unclear. The anti-obesity effects of ST in in vitro and in vivo models, as well as the underlying mechanisms were investigated in this work. We hypothesized that a 70% ethanol extract of S. thunbergii would attenuate obesity by lowering adipogenesis and lipogenesis, inducing browning, and changing the gut microbiota composition in in vitro and in vivo obese models. HPLC and LC-MS data revealed the presence of Indole-2-carboxaldehyde (MF: C9H7NO, MW: 145.16, calcd m/z: 146.06) as one of the bioactive compounds in S. thunbergii extract. In addition, the bioactive substances, p-Coumaric acid, Cinnamic acid, L-rhamnulose, Dulcitol, Loliolide, and Isovalerycarnitine were found in S. thunbergii. In vitro assay, to determine the cytotoxicity of S. thunbergii (ST) and the indicator component indole-2-carboxaldehyde (I2C), a cell viability test was performed in 3T3-L1 preadipocytes. ST and I2C demonstrated greater than 80% cell viability at concentration less than 20 mg/L. Consequently, a continuous experiment was conducted with a 20 mg/L concentration. In addition, the evaluation of the anti-lipogenesis effect revealed that ST and I2C reduced Oil Red O staining at 10 and 20 mg/L compared to the PBS-treated control group. To figure out the anti-adipogenic mechanism of ST, we analyzed gene expressions associated with adipogenesis, lipogenesis, and thermogenesis. ST treatment significantly reduced adipogenesis-related genes (C/ebpα, Pparγ), and increasedmetabolic sensors (Ampk, Sirt1), thermogenic genes (Pgc-1a, Ucp1) and proteins (pAMPK/AMPK and UCP1). In vivo assay, mice were fed a standard chow diet, a high-fat diet (HF), or a HF supplemented with ST (at dosages of 150 and 300 mg/kg b.w. per mouse per day) for eight weeks (n=10/group). Compared to the HF group, ST treatment is more effective at reducing body weight and fat. The area of adipocytes in the epididymal white adipose tissue was 2.2 times greater in the HF group than in the Con group, whereas STtreatment groups (150 and 300 mg/kg bw) significantly reduced the size of adipocytes (36% and 53%, respectively) compared to the HF group. In the liver, the HF group had a greater number of ballooned hepatocytes than the Con group, but the ST treatment groups had fewer ballooned hepatocytes than the HF group. Compared to the HF group, the ST high concentration group (300 mg/kg bw) lowered LDL cholesterol and serum leptin levels, while improving glucose intolerance. The expression of genes Ampk and Sirt1 increased in fat (121% and 241%, respectively) and liver (1044% and 252%, respectively) in the ST high concentration group compared to the HF group. Furthermore, when compared to the HF group, both ST treatments raised the ratio of pAMPK/AMPK. We examined the effects of a high dose of ST (300 mg/kg bw) on the fecal microbiota of obese mice. In reaction to a highfat diet, bacterial diversity was reduced considerably, and bacterial distribution was also altered in compared to the control. The ratio of Firmicutes to Bacteroidetes indicates the instability of the gut microbiota in obese individuals, and this ratio was also induced by a high fat diet. S. thunbergii treatment resulted in a minor improvement in the gut microbiota alterations generated by a high-fat diet, particularly in bacterial alteration at the species level. Bacteroides vulgatus and Faecalibacterium prausnitzii increased in response to ST administration, although Romboutsia ilealis decreased compared to HF-fed mice. These findings suggest that ST treatment could prevent obesity in mice raised to be obese by HF via modulating AMPK activation and gut microbiota composition, and may be utilized as an anti-obesity agent.