In this study, an exopolysaccharide (EPS) named EPS-RB was produced when the gene cluster ycjD-fabl-yciW-rnb were overexpressed in E. coli. Monosaccharide composition analysis revealed that EPS-RB is a novel EPS that consisted of L-fucose, L-arabinose, D-galactose/N-acetyl glucosamine, D-glucose, D-xylose, D-ribose, and D-glucuronic add, and their molecular ratio was approximately 80:3:53:69:1:2:64. The content of carbohydrates, protein, and uronic adds in EPS-RB was 90.35 +/- 135%, 2.62 +/- 0.05% and 8.16 +/- 1.00%, respectively. The interaction between EPS-RB and gut microbiota was investigated using an in vitro batch fermentation system. The results showed that -96% of EPS-RB can be degraded by human fecal microbiota after 72 h fermentation, but few can be degraded by mouse cecal microbiota. Furthermore, high-throughput sequencing showed that EPS-RB regulates the human gut microbiota. The genera Collinsella, Butyricimonas, and Hafnia were enriched in group VIR (EPS-RB as a carbon source) when compared with group VI (no carbon source) and VIS (starch as a carbon source). Short-chain fatty acids (SCFAs) production analysis showed that their concentration was significantly higher in group VIR than groups VI and VIS after 72 h fermentation. In summary, an EPS-RB in E. coli was isolated and its regulatory function on gut microbiota was analyzed. (C) 2019 Elsevier B.V. All rights reserved.