β-Glucan as a component of grain cell walls is consumed daily. However, little is known about whether β-glucan is influenced by the gastrointestinal environment. In this study, we aim to investigate the integrated metabolic process of cereal β-glucan. In vitro simulated digestion and fermentation combined with microbiome and metabolome analysis were used to profile the metabolism of β-glucan. Intriguingly, we found that β-glucan was not hydrolyzed by digestive enzymes but partially degraded by gastric acid environment during in vitro digestion. Moreover, β-glucan was utilized by gut microbiota to produce acetate, propionate and butyrate, concurrently, the relative abundance of Lactobacillus significantly increased and Escherichia-Shigella significantly decreased. The correlation analysis between metabolomics datasets and microorganisms revealed that β-glucan catabolism was also accompanied by amino acid catabolism and linoleic acid biosynthesis. Our study offered a forceful basis for the further exploration of the role of β-glucan and gut microbiota in host health.