Objective To investigate the effects of mannanoligosaccharides (MOS) on the in vitro fermentation characteristics and composition of intestinal microbiota in weaned piglets by using an in vitro microbial fermentation technique, with fructooligosaccharides (FOS) taken as the control. Methods Using microbial inocula derived from the jejunal and colonic chyme of piglets, with FOS and MOS as respective substrates, this study measured microbial gas production and fermentation broth pH at five time points (0, 6, 12, 24, and 48 h), and collected fermentation broth samples at each time point for subsequent microbial analysis. Results In the in vitro jejunal microbial fermentation system, both the fermentation broth pH and gas production in the MOS group were significantly higher than those in the FOS group (P<0.05). At 24 h of fermentation, compared with FOS, MOS significantly increased the concentrations of acetate, propionate, and total short-chain fatty acids (SCFAs) (P<0.01). The formate production in the MOS group was significantly lower than that in the FOS group (P<0.01). At 48 h of fermentation, the lactate concentration in the MOS group was significantly lower than that in the FOS group (P<0.01). In the in vitro colonic microbial fermentation system, gas production in the MOS group was significantly higher than that in the FOS group (P<0.05). At 48 h of fermentation, the production of formate, acetate, butyrate, SCFAs, and lactate in the MOS group was significantly higher than that in the FOS group (P<0.01). The 16S rRNA gene sequencing results of the jejunal fermentation broth revealed that at 48 h of fermentation, both the Shannon and Simpson indices in the MOS group were significantly higher than those in the FOS group (P<0.01). Furthermore, the microbiota composition exhibited disparity between the MOS and FOS groups. Moreover, the relative abundances of Bifidobacterium, Limosilactobacillus, and Megasphaera were significantly higher in the MOS group than in the FOS group (P<0.05). Conclusion Compared to FOS, MOS enabled the microbiota in the small intestine of piglets to significantly improve the microbial community structure, increase the abundance of beneficial bacteria such as Bifidobacterium, enhance gas production, and promote the generation of acetate and other SCFAs. These findings suggested that MOS held potential for modulating microecology in the small intestine of weaned piglets.