Oxidized cholesterol (OXC) is a harmful dietary substance. Although the consumption of OXC has been associated with colonic inflammation, related underlying mechanisms are still limited. We evaluated the influence of dietary OXC on gut health and ecology by applying the murine model. Results showed that the thickness of the mucus layer was significantly reduced in healthy mice treated with OXC. Short-term intake of OXC did not influence the expression of pro-inflammatory factors in healthy mice but it induced the decrease of Muc2 expression in the proximal colon, accompanied by an increase in the abundance of 2 mucus-degrading bacteria, namely Akkermansia muciniphila and Bacteroides acidifaciens. Consistently, oral exposure of OXC promoted mucus barrier erosion in dextran sulfate sodium (DSS)-induced colitis mice and facilitated bacteria infiltration in the colon. The adverse effect of OXC on mucus layer disappeared in antibiotics-treated healthy mice, suggesting that the damaging effect of OXC on the gut mucus layer was not direct and instead was mediated by causing microbiota dysbiosis. Finally, the impact of OXC on the mucus layer and colitis was partly alleviated by green tea catechins. These studies demonstrated that the OXC-induced mucus barrier damage was mainly induced by the dysregulation of gut microbiota at least in this mouse model.