Abstract: Functional oligosaccharides can be categorized into long-chain oligosaccharides and short-chain oligosaccharides based on the complexity of their chemical structures, and specific combinations of the two types exert distinctive biological effects. In this study, inulin and stachyose were co-administered to the gut microbiota of healthy human subjects. The optimal combination ratio of inulin to stachyose was screened using gas production volume as the key indicator. Subsequently, an in vitro gut microbiota fermentation experiment was conducted. The dynamic changes in gas production and short-chain fatty acids (SCFAs) levels over time were monitored, and alterations in the composition of the gut microbiota were analyzed via 16S rRNA gene sequencing, aiming to explore the fermentation characteristics of the combined oligosaccharides. The findings indicated that the mixture with an inulin-to-stachyose ratio of 7:1 exhibited the minimum gas production, which was significantly reduced by 26% compared with the inulin-only group (P<0.05). Meanwhile, the total SCFA content of the combined treatment group was markedly lower than that of the inulin single group and stachyose single group (P<0.05). The combined application of inulin and stachyose exerted a significant proliferative effect on both Lactobacillus and Bifidobacterium. Notably, the addition of stachyose impaired the utilization of inulin by the family Lachnospiraceae, thereby reducing the gas production during fermentation. This study concluded that the combined use of inulin and stachyose could reduce gas production in vitro fermentation, slow down the gas production rate, and also had good probiotic effects, which could provide certain guidance for the development of inulin-stachyose combination products.