Two publicly available oat (Avena sativa) lines, “Jim” and “Paul” (5.17 and 5.31% β-glucan, respectively), and one experimental oat line “N979” (7.70% β-glucan), were used to study the effect of β-glucan levels in oat flours during simulated in vitro digestion and fermentation with human fecal flora obtained from different individuals. The oat flours were digested by using human digestion enzymes and fermented by batch fermentation under anaerobic conditions for 24 h. The fermentation progress was monitored by measuring pH, total gas, and short-chain fatty acid (SCFA) production. Significant effects of β-glucan on the formation of gas and total SCFA were observed compared to the blank without substrate (P < 0.05); however, there were no differences in pH changes, total gas, and total SCFA production among oat lines (P > 0.05). Acetate, propionate, and butyrate were the main SCFA produced from digested oat flours during fermentation. More propionate and less acetate were produced from digested oat flours compared to lactulose. Different human fecal floras obtained from three healthy individuals had similar patterns in the change of pH and the production of gas during fermentation. Total SCFA after 24 h of fermentation were not different, but the formation rates of total SCFA differed between individuals. In vitro fermentation of digested oat flours with β-glucan could provide favorable environmental conditions for the colon and these findings, thus, will help in developing oat-based food products with desirable health benefits.