Titanium dioxide nanoparticles (TiO₂-NPs) are commonly used as food additives, including some high-fat foods that are risk factors for obesity. However, little is known about the effects of chronic TiO₂-NPs digestion in the population on high fat diet (HFD). Herein, we reported that TiO₂-NPs exacerbated HFD-induced obesity by disruption of mucus layer and alterations of gut microbiota. Oral intake of TiO₂-NPs significantly increased body weight, liver weight, and amount of adipose tissues, especially in HFD-fed mice. Mechanistic studies revealed TiO₂-NPs induced colonic mucus layer disruption and obesity-related microbiota dysbiosis. The damage on mucus was demonstrated through down-regulation of Muc2 gene and the absorption of mucin protein by TiO₂-NPs. Consequently, mucus layer damage combined microbiota dysbiosis escalated the low-grade systemic inflammation, which exacerbated HFD-induced obesity. In contrast, gut microbiota depletion eliminated these effects, indicating gut microbiota were necessary for TiO₂-NPs-induced inflammation and obesity. All the results stated the alarming role of TiO₂-NPs in the HFD-driven obesity and emphasized the reevaluating the health impacts of nanoparticles commonly used in daily life, particularly, in susceptible population.