Pulque is a traditional Mexican fermented beverage that harbors a diverse microbial community with potential probiotic properties. However, its functional impact in animal models remains underexplored. This study aimed to evaluate the effects of pulque-derived microbiota on host metabolism and gut microbial composition in a high-fat diet (HFD) mouse model. C57BL/6 mice were divided into groups fed either a normal diet or an HFD, with or without pulque microbiota supplementation for 8 weeks. Metabolic parameters such as glucose tolerance, insulin resistance, and body weight were assessed, along with gut microbiota diversity and composition through 16S rRNA gene sequencing. Results indicated that pulque microbiota exerted a protective effect by promoting the persistence of beneficial families such as Lactobacillaceae and Akkermansiaceae, while moderating shifts in Lachnospiraceae, a family associated with inflammation. Moreover, it attenuated HFD-induced metabolic impairments. In conclusion, pulque-derived microbiota demonstrated a potential probiotic effect by improving metabolic outcomes, limiting the expansion of dysbiosis-associated taxa, and enhancing the presence of beneficial microbial groups. These findings support its potential use in dietary strategies aimed at preventing metabolic disorders related to high-calorie diets.