The fibroblast cell line of 3T3-L1 was used as a cell model for screening and evaluating the feasibility of probiotic components in improving animal lipid metabolisms. The extracts from 12 Lactobacillus strains caused significantly reduced triacylglycerol (TAG) accumulation but with severe inflammation induction in 3T3-L1 adipocytes. Interestingly, exopolysaccharides (EPS) from LGG (Lactobacillus rhamnosus GG) significantly decreased the TAG accumulation without any inflammation. The anti-obesity effect of EPS was confirmed in high-fat-diets feeding mice. Fat pads of mice injected with EPS (50 mg/kg) every two days for two weeks were significantly reduced with much smaller adipocytes, compared with the counterparts. The levels of TAG and cholesterol ester in liver, as well as serum TAG, were decreased in EPS injected mice. In addition, down-regulated inflammation was observed in adipose tissue and liver. Interestingly, the expression of TLR2 in adipose tissue and 3T3-L1 cells was significantly increased by EPS addition. Moreover, the reverse of TAG accumulation in TLR2 knockdown 3T3-L1 in the presence of EPS confirmed that the inhibition effect of EPS on adipogenesis was mediated by TLR2. EPS from LGG has the potential for therapeutic development to intervene lipid metabolic disorders in mammals.