Glucosylceramide is present in many foods, such as crops and fermented foods. Most glucosylceramides are not degraded or absorbed in the small intestine and pass through the large intestine. Glucosylceramide exerts versatile effects on colon tumorigenesis, skin moisture, cholesterol metabolism and improvement of intestinal microbes in vivo. However, the mechanism of action has not yet been fully elucidated. To gain insight into the effect of glucosylceramide on intestinal microbes, glucosylceramide was anaerobically incubated with the dominant intestinal microbe, Blautia coccoides, and model intestinal microbes. The metabolites of the cultured broth supplemented with glucosylceramide were significantly different from those of broth not treated with glucosylceramide. The number of Gram-positive bacteria was significantly increased upon the addition of glucosylceramide compared to that in the control. Glucosylceramide endows intestinal microbes with tolerance to secondary bile acid. These results first demonstrated that glucosylceramide plays a role in the modification of intestinal microbes.