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1-Butyl-3-methylimidazolium chloride (BmimCl), an imidazolium-based ionic liquid, is considered the representative emerging persistent aquatic pollutant, and its environmental toxicity has attracted a growing concern. However, most of the investigations focused on monocultures or a single organism, with little information available on the complex syntrophic consortium that dominates the complex and successional biochemical processes, such as anaerobic digestion. In this study, the effect of BmimCl at environmentally relevant levels on glucose anaerobic digestion was therefore investigated in several laboratory-scale mesophilic anaerobic digesters to provide such support. Experimental results showed that BmimCl at 1-20 mg/L inhibited the methane production rate by 3.50-31.03%, and 20 mg/L BmimCl inhibited butyrate, hydrogen, and acetate biotransformation by 14.29%, 36.36%, and 11.57%, respectively. Toxicological mechanism studies revealed that extracellular polymeric substances (EPSs) adsorbed and accumulated BmimCl through carboxyl, amino, and hydroxyl groups, which destroyed the EPSs' conformational structure, thereby leading to the inactivation of microbial cells. MiSeq sequencing data indicated that the abundance of Clostridium_sensu_stricto_1 , Bacteroides , and Methanothrix decreased by 6.01%, 7.02%, and 18.45%, respectively, in response to 20 mg/L BmimCl. Molecular ecological network analysis showed that compared with the control, the lower network complexity, fewer keystone taxa, and fewer associations among microbial taxa were found in the BmimCl-present digester, indicating the reduced stability of the microbial community.