Titanium dioxide nanoparticles (TiONPs) have been widely used as food additives in daily life. However, the impact of oral intake of TiONPs on the nervous system is largely unknown. In this study, 7-week-old mice were treated with either vehicle or TiONPs suspension solution at 150 mg/kg by intragastric administration for 30 days. Our results demonstrated that oral exposure to TiONPs resulted in aberrant excitement of enteric neurons, although unapparent pathological changes were observed in gut. We also found the richness and evenness of gut microbiota were remarkably decreased and the gut microbial community compositions were significantly changed in the TiONP-treated group as compared with vehicle controls. Interestingly, oral exposure to TiONPs was capable to induce the inhibitory effects on locomotor activity, but it did not lead to significant change on the spatial learning and memory ability. We further revealed the mechanism that TiONPs could specifically cause locomotor dysfunction by elevating the excitement of enteric neuron, which might spread to brain via gut-brain communication by vagal pathway. However, inflammation response, enteric neurotransmitter 5-HT and major gut peptides might not be involved in this pathological process. Together, these findings provide valuable insights into the novel mechanism of TiONP-induced neurotoxicity. Understanding the microbiota-gut-brain axis will provide the foundation for potential therapeutic or prevention approaches against TiONP-induced gut and brain-related disorders.