In this study, konjac glucomannan (KGM) was acetylated, followed by the investigation of its corresponding structural and physicochemical properties. Spectroscopic analyses, including FTIR and NMR, confirmed the introduction of acetyl groups. Rheological measurements found a decrease in both viscosity and gel strength of the acetylated KGM (AKGM), attributing to weaker intermolecular interactions. The current study also revealed that the acetylation of KGM led to reduced gut microbiota fermentation kinetics, followed by a lower short-chain fatty acids (SCFAs) generation, which may be due to the stearic hindrance raised from the introduction of acetyl groups onto its macromolecules. Interesting, although both two types of polysaccharides (before and after acetylation) improved gut microbiota profile in term of enriched probiotics profile, AKGM even generated a lower ratio of Firmicutes/Bacteroidota compared to that of KGM, which was negatively correlated with the degree of acetylation of KGM. Importantly, this was the first time to reveal that Prevotella_9 was selectively promoted in AKGM, followed by a greatly depressed growth of pathogens, such as Escherichia-Shigella compared to KGM. Nevertheless, this study demonstrated that the change in the molecular structure of the polysaccharides targets the promotion of specific bacteria, which may highlight their potent application for individual healthy intervention.