The 2-ethylhexyl diphenyl phosphate (EHDPHP), a primary organophosphorus flame retardant used in various industrial products, is prone to biotransformation. However, there is a knowledge gap on the sex- and tissue-specific accumulation and potential toxicities of EHDPHP (M1) and its metabolites (M2-M16). In this study, adult zebrafish (Danio rerio) were exposed to EHDPHP (0, 5, 35 and 245 µg/L) for 21-day, which was followed by 7-day depuration. The bioconcentration factor (BCF) of EHDPHP in female zebrafish was 26.2 ± 7.7% lower than in males due to the lower uptake rate (k_u) while higher depuration rate (k_d) in the females. The regular ovulation and higher metabolic efficiency promoted elimination from female zebrafish, thus leading to much less (28–44%) accumulation of ∑(M1-M16) in female zebrafish. They exhibited the highest accumulation in the liver and intestine in both sexes, which might be regulated by tissue-specific transporters and histones evidenced by molecular docking results. Intestine microbiota analysis further revealed that female zebrafish were more susceptible to EHDPHP exposure, with more significant changes in phenotype number and KEGG pathways in female than male fish. Disease prediction results suggested that EHDPHP exposure might cause cancers, cardiovascular diseases as well as endocrine disorders in both sexes. These results provide a comprehensive understanding of the sex-dependent accumulation and toxicity of EHDPHP and its metabolites.