Imidacloprid enters the water environment through rainfall and causes harm to aquatic crustaceans. However, the potential chronic toxicity mechanism of imidacloprid in crayfish has not been comprehensively studied. In this study, red claw crayfish (Cherax quadricarinatus) were exposed to 11.76, 35.27, or 88.17 μg/L imidacloprid for 30 days, and changes in the physiology and biochemistry, gut microbiota, and transcriptome of C. quadricarinatus and the interaction between imidacloprid, gut microbiota, and genes were studied. Imidacloprid induced oxidative stress and decreased growth performance in crayfish. Imidacloprid exposure caused hepatopancreas damage and decreased serum immune enzyme activity. Hepatopancreatic and plasma acetylcholine decreased significantly in the 88.17 μg/L group. Imidacloprid reduced the diversity of the intestinal flora, increased the abundance of harmful flora, and disrupted the microbiota function. Transcriptomic analysis showed that the number of up-and-down-regulated differentially expressed genes (DEGs) increased significantly with increasing concentrations of imidacloprid. DEG enrichment analyses indicated that imidacloprid inhibits neurotransmitter transduction and immune responses and disrupts energy metabolic processes. Crayfish could alleviate imidacloprid stress by regulating antioxidant and detoxification-related genes. A high correlation was revealed between GST, HSPA1s, and HSP90 and the composition of gut microorganisms in crayfish under imidacloprid stress. This study highlights the negative effects and provides detailed sequencing data from transcriptome and gut microbiota to enhance our understanding of the molecular toxicity of imidacloprid in crustaceans. Display omitted •Imidacloprid strongly inhibited the growth performance of Cherax quadricarinatus.•Imidacloprid suppressed immunity and caused hepatopancreatic damage in C. quadricarinatus.•Imidacloprid reduces the diversity and destroys the balance in the intestinal flora of crayfish.•Imidacloprid may alter gene expression by affecting intestinal flora.