The gut microbiome can be readily influenced by external factors, such as nanomaterials. However, the role of the microbiota-gut-brain axis in nanomaterials-induced neurotoxicity remains largely unknown. In this study, young mice aged 4 weeks were treated with either a vehicle solution or 26 mg kg-1 zinc oxide nanoparticles (ZnONPs) by intragastric administration for 30 days. The neurobehavioral alterations were assessed by the Morris water maze and open field test. Gut microbiota and the metabolites in both blood and hippocampus were detected using 16S rRNA sequencing and liquid chromatography-mass spectrometry metabolomics, respectively. The results demonstrated that oral exposure to ZnONPs resulted in neurobehavioral impairments in young mice, mainly manifested by spatial learning and memory deficits, and the inhibition of locomotor activity. Intriguingly, ZnONPs caused a marked disturbance of the gut microbial composition, but did not alter the α-diversity of the microbiota. The correlation analysis further revealed that neurobehavioral impairments induced by ZnONPs were closely associated with a perturbation in the gut microbiota composition that were specific to changes of neurobehavior-related genes (such as Bdnf and Dlg4), and correlated with serum and hippocampal metabolites. We also identified a unique metabolite [DG(15:0/0:0/22:4n6)] that linked relationships among the gut microbiota, metabolites and neurobehavior-related genes. Taken together, our results illustrated that oral exposure to ZnONPs not only altered the gut microbiome community, but also substantially disturbed the metabolic profiles leading to neurobehavioral impairments via the microbiota-gut-brain axis. These findings will provide a novel view for understanding the neurotoxicity of ZnONPs, and are helpful for identifying potential prevention and treatment strategies.