Abstract Obesity affects more than one half of the global population. This study aims to evaluate the role of soybean powder fermented by Bacillus subtilis SJLH001 (FSP) in the prevention of obesity and the related mechanisms. FSP exhibited higher antioxidant activity, fibrinogen degradation, and anticoagulating activity in vitro. In vivo tests involved in a high-fat induced obesity model revealed that FSP supplementation resulted in lower triglyceride level, reduced fat mass, and smaller adipocyte size in mice. FSP also ameliorated abnormal physiology of ileum, colon and kidney, decreased uric acid and creatinine levels of perirenal fat, and increased abundance of beneficial fecal bacteria based on culturomics analysis. Gut transcriptomic analysis revealed that transport processes (cation, anion, and lipid transport), angiogenesis (arachidonic acid metabolism and angiogenesis factor), inflammation, neuronal signaling, mucus integrity, and IGF signaling were involved in reducing adiposity by FSP. These results suggested that FSP could be used as an anti-obesity probiotic food in mice.