Given the probiotic effects previously found in subsp. MN-Gup (MN-Gup) and its great application potential in dairy products, this study aimed to investigate the effects of fermented milk containing MN-Gup or MN-Gup-based synbiotics on high fat diet (HFD)-induced obesity in rats. Galacto-oligosaccharides (GOS) and xylo-oligosaccharides (XOS) were selected as the tested prebiotics in MN-Gup-based synbiotics due to their promotion of MN-Gup growth in vitro. After nine weeks of HFD feeding, the obese rats were intervened with fermented milk containing MN-Gup (MN-Gup FM) or its synbiotics (MN-Gup + GOS FM, MN-Gup + XOS FM) for eight weeks. The results showed that the interventions could alleviate HFD-induced body weight gain, epididymal fat deposition, adipocyte hypertrophy, dyslipidemia and inflammation, but GOS and XOS did not exhibit significant synergies with MN-Gup on those alleviations. Furthermore, the interventions could regulate the HFD-affected gut microbiota and microbial metabolites, as shown by the increases in short chain fatty acids (SCFAs) and alterations in obesity-related bile acids (BAs), which may play important roles in the mechanism underlying the alleviation of obesity. This study revealed the probiotic effects of MN-Gup on alleviating obesity and provided the basis for MN-Gup applications in the future.