Among plasticizers, dibutyl phthalate (DBP) is widely used in in industry, posing significant health risks to aquatic organisms. In this study, adult male and female zebrafish were exposed to 0 and 30 μg/L DBP for 15 days. Behavioral monitoring, immunofluorescence, protein immunoblotting, and high-throughput sequencing were used to investigate the critical role of the gut microbiome in DBP-induced dysfunction of the zebrafish gut-brain axis. The results showed pronounced, sex-specific toxic effects of acute DBP exposure in adult zebrafish, with males experiencing more severe neurological damage, while females exhibited greater intestinal damage. DBP exposure caused marked anxiety behaviors in males and significant weight loss in females. Males showed reduced neuronal expression, while females exhibited increased intestinal permeability and lower levels of the tight junction protein (ZO-1). The Firmicutes/Bacteroidota (F/B) ratio decreased, indicating severe gut microbiota dysbiosis. Changes in the gut and fecal microbiota composition, along with PICRUSt2 functional predictions, suggest that female zebrafish experienced more severe metabolic disturbances than males. Analysis of key gene expression in the brain-derived neurotrophic factor (bdnf) pathway revealed that changes in the abundance of tryptophan-metabolizing bacteria in the gut may explain the sex-specific effects of DBP on neurotransmitter serotonin levels in the brain, which influence the gut-brain axis in zebrafish. This study contributes to the understanding of toxic effects of DBP on aquatic organisms and provides strong evidence for assessing its environmental risks.