Diethyl phthalate (DEP) had widely presented in various industrial wastewater, and caused negative effect on activated sludge. However, the responses of activated sludge involving microstructure and bacterial community induced by DEP are not well explored. Hence response mechanism of activated sludge with DEP exposure was explored combining sequencing batch reactor and Illumina MiSeq sequencing approaches. Results showed DEP over 50 mg/L was toxic to microorganism and impeded activated sludge removing COD. Activated sludge became loose and porous, and filamentous bacteria appeared due to the toxicity of DEP (over 100 mg/L). Furthermore, DEP exposure led to an overall increase in EPS from 102.1 mg/g MLVSS at stage DEP (0) to 274.8 mg/g MLVSS at stage DEP (150), suggesting EPS was probably one of the detoxification mechanisms. Illumina MiSeq sequencing approaches again showed DEP was poisonous to microorganism in form of decrease in the richness and evenness of the bacterial community and shifts in community structure. Whereas, an increase in the abundance of DEP-degrading genera, like Acinetobacter and Chryseobacterium, , was observed under DEP exposure, which was another detoxification mechanism. In addition, the relative abundance of metabolic pathways involved in carbon metabolism and DEP degradation increased to 10.80 % and 2.07 % at stage DEP (150), respectively, which was also important in combating DEP exposure.