Mycotoxin and pesticide co-contamination represents a critical threat to aquatic ecosystem integrity, yet the toxicological effects and underlying mechanisms of T-2 toxin (T-2) and diniconazole (DIN), two widely coexisting agricultural contaminants, remain elusive. Here, adult male and female zebrafish were exposed to T-2 (4.25 μg L⁻¹), DIN (32.79 μg L⁻¹), or their combination for 60 days to investigate gut-liver homeostasis dysregulation and related toxic effects. Results demonstrated that co-exposure significantly perturbed gut microbial community structure and disrupted gut-liver balance. This was accompanied by a positive correlation between intestinal and hepatic lipopolysaccharide (LPS) concentrations. Co-exposure markedly increased hepatic reactive oxygen species (ROS), lipid peroxidation (LPO) levels and catalase (CAT) activity. T-2 and DIN disrupted iron metabolism and ferroptosis-related responses, characterized by increased Fe 2 + levels, altered expression of iron metabolism-related genes such as transferrin ( tf ), transferrin receptor ( tfr ), and ferritin heavy chain ( fth ), reduced the solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) protein abundance, and decreased GPX4 activity. These results indicate that oxidative stress-associated ferroptotic processes may represent an important mechanism underlying hepatic toxicity induced by combined T-2 and DIN exposure. Sex differences were observed. Females exhibited more severe intestinal barrier damage and inflammation under single exposure, while males showed greater overall toxic impairment under co-exposure. These findings enhance our understanding of the combined toxicity of mycotoxin-pesticide co-contamination and provide mechanistic insights into potential ecological impacts in aquatic organisms.