The prevalence of obesity has increased and is a health concern worldwide. Due to the concerns regarding synthetic anti-obesity treatments, nowadays natural products become a trend. Previous studies proved that there is a potential to use marine algae as anti-obesity agents. Therefore, in this study, the lipid inhibitory effect of crude polysaccharide of amyloglucosidase-assisted hydrolysate from Sargassum thunbergii (STAC) and its fucoidan fractions (STAFs) on 3T3-L1 cells and high-fat diet (HFD)-induced obese mice were investigated. According to the results, the STAF3, showed the highest xylose content and exhibited significant inhibitory effects on lipid accumulation by downregulating adipogenic and lipogenic proteins in 3T3-L1 cells. Furthermore, oral supplementation with STAC significantly declined gain in body weight and fat weight, and serum lipid contents in an HFD-induced obesity mouse model. Structural and chemical characterizations demonstrated that purified STAF3 has consistent surface morphology and small particle size, with similar structural characteristics as commercial fucoidan. Together, these results indicate that STAC and purified STAF3 from Sargassum thunbergia is a potent source to develop as ananti-obesity agents or functional food products to counter obesity.