Objective: To evaluate the effects of mechanical processing on the physicochemical properties of oat β-glucan and its in vitro fermentation. Methods: Oat coarse grains, oat flakes, and oat flour were respectively produced through mechanical progressing methods such as steel cutting, table pressing, and grinding. Then, scanning electron microscopy was used to observe the microstructure, the content, dissolution rate and relative molecular weight of oat β-glucan were determined, and the colon environment was simulated in vitro for fermentation. Results: The total β-glucan content and dissolution rate of steel-cutting oat were higher than those of untreated sample. The total β-glucan content of oat flake and oat flour were lower than untreated oat, but the dissolution rate was higher than it. Untreated oat showed a lower fermentation rate, with a significantly lower acid and gas production rate than those of steel-cutting oat, oat flake, and oat flour. The total short chain fatty acid content of untreated oat produced by fermentation was the lowest, but the content of propionic acid and butyric acid was significantly higher than the other three groups. Conclusion: Mechanical progressing can affect the in vitro fermentation characteristics of oats by altering the integrity of their cell wall structure, as well as the content and dissolution rate of β-glucan. Moderate processing can contribute to the health benefits of oats.