This study investigated the effects of Lacticaseibacillus paracasei K56 (L. paracasei K56) on body weight, body composition, and glycolipid metabolism in mice with high-fat diet-induced obesity and explored the underlying mechanisms. Male C57BL/6J mice were fed a high-fat diet for 8 weeks to induce obesity; then, the obese mice were gavaged with or without L. paracasei K56 for 10 weeks. The body weight, body composition, fat mass, blood lipid, blood glucose, and hormones of the mice were evaluated. Moreover, the fatty acid synthesis (FAS) and peroxisome proliferator-activated receptor γ (PPAR-γ) expressions in the liver were detected via Western blotting. 16S rRNA gene sequencing was adopted to determine the gut microbiota alterations. The high-fat diet successfully induced obesity, as indicated by the abnormal increase in body weight, visceral fat, fat mass, blood lipids, fasting blood glucose, and insulin-resistance. Moreover, the FAS expression in the liver was significantly increased, whereas the PPAR-γ expression was significantly decreased. The relative abundance of Proteobacteria, Actinobacteria and Patescibacteria was also significantly increased, and that of Verrucomicrobia was significantly decreased. However, these indicators of mice supplemented with L. paracasei K56 were significantly opposite to those of obese mice. The Ruminococcuaceae_UCG-013, Akkermansia, Prevotellaceae_UCG-001, Muribaculum, and Lachnospiraceae_NK4A136 groups were significantly negatively correlated with body weight, blood lipids, and blood glucose-related indicators, whereas Coriobacteriaceae_UCG-002, Enterorhabdus, Raoultibacter, Acinetobacter, Romboutsia, Leuconostoc, and Erysipelatoclostridium were significantly positively correlated with these indicators. L. paracasei K56 might be a promising probiotic strain that could effectively slow down the body weight gain, reduce fat accumulation, alleviate insulin-resistance, and restore pancreatic β-cell function in obese mice by regulating the gut microbiota. © 2022. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.