Imidacloprid (IMI) is used in integrated aquaculture systems for pest control and the toxicity of IMI to non-target aquatic animals such as fish and microcrustaceans has been recognised. However, knowledge about the toxic effect of IMI on commercial crabs is still scarce. In the present study, effects of IMI on the acute toxicity, antioxidative status, detoxification systems and gut microbiota in Chinese mitten crab, Erocheir sinensis were investigated. In the present study, the 96-h LC of IMI for E. sinensis was 24.97 mg/L. Under sublethal exposure, superoxide dismutase (SOD) activities increased under low concentration (LC, 5 μg/L) and median concentration (MC, 50 μg/L) exposure, but decreased in high concentration group (HC, 500 μg/L). Activities of catalyse (CAT) decreased in a dose-dependent manner. Detoxification-related enzymes aminopyrine N-demethylase (APND) and erythromycin N-demethylase (ERND) increased in all treatments whereas glutathione-S-transferase (GST) decreased dose-dependently. The relative mRNA expression of the cytochrome P4502 (cyp2) gene was induced significantly in LC and HC groups while no significant change was observed in cytochrome P4503 (cyp3) gene. The expression of gst was also significantly decreased in HC group. Up-regulation of heat shock protein hsp70 and 90 was observed in MC and HC groups whereas hsp60 up-regulated only in LC group. In addition, significant changes of composition of microbial communities at both phylum and genus levels were found in this test. In particular, beneficial bacteria were found to decrease and pathogens increased after exposure to IMI. These results indicate that high concentration of IMI could induce oxidative stress and suppress the detoxification system mainly by down-regulation of gst mRNA expression, inhibition of enzyme activities and dysbiosis of gut microbiota.