Oat β-glucan (OG) is a common food in many diets, but the relationship between the structural changes of dietary fibre and the dynamic shifts in gut microbiota composition remains unclear. In this study, the dynamic variations of physicochemical and structural characteristics of OG at different fermentation stages were studied using an in vitro porcine colonic digestion model. lMeanwhile, the correlation between the molecular structure changes of OG and its regulation of gut microorganisms during fermentation was studied. The molecular weight of OG decreased with the prolongation of fermentation time due to the decomposition of the glucoside bond by gut microorganisms. Methylation analysis showed that the sugar residues in OG mainly included T-Glcp, 3-Glcp and 4-Glcp, while the proportions of 3-Glcp and 4-Glcp did not change significantly during the process of fermentation, demonstrating that gut microorganisms could equally decompose β-(1, 3) and β-(1, 4) glucoside bonds. During the fermentation process, OG inhibited the growth of harmful bacteria while promoting the growth of beneficial bacteria, especially Lactobacillus. Compared with gum arabic, hawthorn pectin, arabinoxylan, guar gum and dendrobium officinale polysaccharide, OG showed the fastest fermentation rate and highest Lactobacillus abundance. This study's results offer a scientific foundation for enhancing the conversion of oat β-glucan (OG) into prebiotics within the functional food industry. Additionally, they provide valuable insights into the reciprocal regulation between other structural polysaccharides and gut microorganisms.