METHODS AND RESULTS: A total of 40 male Sprague-Dawley rats were assigned to one of five groups of varying diets as follows: standard diet, high fat diet (HFD), HFD supplemented with Lactobacillus casei strain Shirota, HFD supplemented with Bifidobacterium longum and HFD supplemented with a mixture of these two bacterial species. After 15 weeks of supplementation, the animals were examined for changes in body weight, body fat, total count of bacteria in fecal, blood serum lipid profile, leptin, adiponectin and inflammatory biomarkers. Histological analysis of the liver and adipose tissue was performed and the hepatic mRNA expression levels of genes related to lipid metabolism were measured. It was found that probiotic supplementation of either B. longum or a mixture of B. longum and LcS bacteria significantly reduced weight and triglycerides in the HFD groups. Supplementation of B. longum bacteria showed better results in terms of modulating leptin level, fat mass, adipocyte size and lipoprotein lipase expression, as well as increasing adiponectin and peroxisome proliferator-activated receptors-γ expression compared to dual species of bacteria. No significant differences were observed in the total count of fecal bacteria, glucose and inflammatory biomarker levels between supplemented groups.