Chlorantraniliprole, a widely used diamide insecticide in rice cultivation, poses ecotoxicological risks to Procambarus clarkii in rice-crayfish integrated systems (RCIS), a prevalent aquaculture practice in China. This study integrates transcriptomics, intestinal microbiota analysis, and physiological assessments to elucidate these effects. We determined a 96 h LC50 of 154.15 mg/L, indicating low acute toxicity. Chronic exposure (14 days) at environmentally relevant (0.05 mg/L) and elevated (5.00 mg/L) concentrations revealed significant bioaccumulation in the hepatopancreas and intestine, leading to dose-dependent histopathological damage, oxidative stress, and disrupted intestinal microbiota. Transcriptomic analysis identified 2294 differentially expressed genes (89.4% downregulated) under high-dose exposure, highlighting suppressed metabolic processes and endoplasmic reticulum stress. Molecular docking confirmed strong binding affinities (ΔG = -33.54 to -66.36 kcal/mol) between chlorantraniliprole and antioxidant enzymes, suggesting direct inhibition mechanisms. These findings provide novel insights into the multilevel toxicity of chlorantraniliprole in crustaceans and establish a foundation for ecological risk assessments in RCIS.