<p>Prometryn, a persistent triazine herbicide widely applied in agriculture and aquaculture, presents ecological and food safety risks due to its environmental persistence and bioaccumulative properties. However, its metabolic fate, tissue kinetics, and effects on intestinal microbiota remain poorly characterized in crustaceans. This study employed an integrated approach to investigate the impacts of prometryn on red swamp crayfish (<i>Procambarus clarkii</i>) via waterborne exposure at 2.88 mg/L (20% of the 96-h LC₅₀). Using high-resolution mass spectrometry-based metabolite identification, residue quantification, non-compartmental pharmacokinetic modeling, and 16S rRNA gene sequencing, we assessed bioaccumulation, biotransformation, and gut microbial dysbiosis. Four major metabolites, including deisopropyl prometryn, prometryn sulfoxide, a sulfoxide isomer, and its carboxylic acid derivative, were identified for the first time in crayfish. Pharmacokinetic analysis revealed substantial bioaccumulation, with the hepatopancreas as the primary accumulation site and prolonged retention (<i>T</i>_1/2 = 27.97 h in males). Notably, prometryn residues in edible muscle reached up to 229.3 µg/kg, raising food safety concerns. Exposure also induced sex-dependent intestinal dysbiosis: males showed reduced alpha diversity, depletion of Firmicutes, and enrichment of Bacteroidetes; females exhibited compositional shifts including elevated Tenericutes, reduced Proteobacteria, and decreased <i>Vibrio</i> abundance, suggesting potential disruption of gut barrier function, nutrient metabolism, and immune homeostasis. Overall, this study elucidates the internal exposure dynamics and sublethal ecotoxicity of prometryn in crayfish, providing critical data to support the refinement of pesticide residue standards and ecological risk assessment in aquaculture environments.</p>