The primary objective was to determine the beneficial effects of oat β-glucan (OG) and barley β-glucan (BG) on gut health. A total of 200 male Sprague-Dawley rats were divided into 5 groups of 40 rats each, control group (CON), low-dose OG-administered group (OGL), high-dose OG-administered group (OGH), low-dose BG-administered group (BGL), and high-dose BG-administered group (BGH). OGL and OGH were administered oat β-glucan by intragastric gavage at a dose of 0.35 g/kg of body weight (BW) and 0.70 g/kg of BW daily for 6 weeks, and BGL and BGH were administered barley β-glucan. The CON received normal saline. Intestinal-health-related indexes were analyzed at baseline, week 3, week 6, and week 7. Cereal β-glucan significantly influenced the fecal water content, pH value, ammonia levels, β-glucuronidase activity, azoreductase activity, and colonic short-chain fatty acid (SCFA) concentrations (p < 0.05). Moreover, the population of Lactobacillus and Bifidobacterium increased (p < 0.05), whereas the number of Enterobacteriaceae decreased (p < 0.05) in a dose-dependent manner during the period of cereal β-glucan administration. These results suggested that cereal β-glucan might exert favorable effects on improving intestinal functions and health but the gut-health-promoting effects of oat β-glucan were better than those of barley β-glucan.