In this research, the potential anti-obesity efficacy ofLactobacillus rhamnosusBST-L.601 and its fermented product (named SPY) with mashed sweet potato paste were investigated using 3T3-L1 preadipocytes and high-fat diet (HD)-induced obese mice. SPY (0–0.5 mg/mL) dose-dependently and significantly reduced lipid accumulation and TG content and the expression of adipogenic markers (C/EBPα, PPAR-γ, and aP2) and fatty acid synthetic pathway proteins (ACC and FAS) in 3T3-L1 adipocytes, demonstrating that SPY suppresses adipocyte differentiation and lipogenesis. Oral administration of SPY (4 × 107CFU/kg body weight) to HD-induced obese mice for 12 weeks significantly reduced the body and liver weight, the size of adipocytes, and the weight of epididymal, visceral, and subcutaneous fat tissues. SPY was more effective in decreasing body weight gain in HD mice than in treatment with BST-L.601 alone. Administration of SPY or BST-L.601 also reduced the serum level of total cholesterol and LDL cholesterol and leptin secretion at a similar level. These results revealed that both SPY and BST-L.601 effectively suppress HD-induced adipogenesis and lipogenesis, suggesting that these materials would be useful in the functional foods industry to ameliorate and/or prevent obesity.