The effects of the influent nitrate (NO3−-N) concentration on reactor performance and microbial communities of anaerobic methane oxidation coupled to denitrification in a membrane biofilm reactor were emphatically investigated after prolonged storage. The system was run for a period of 185 days including the storage period, reactivating process, and stable operation phase. The influent NO3−-N concentrations varied from 10, 20, and 30 mg/L, and the reactor was continuously operated for 135 days. The denitrification rate in the reactor was activated first and then inhibited, with the average value of NO3−-N consumed of 0.42 mg/L·h−1 at influent NO3−-N of 20 mg/L; there was no obvious nitrite and ammonia–nitrogen accumulation in the whole stage. Illumina MiSeq sequencing results showed that increases in NO3−-N loading could improve the microbial abundance in the system. The highest value for variety and the lowest value for the evenness of the microbial community corresponded to the ideal value of 20 mg/L. It speculated that promoting biofilm bacteria (PBB) became the key functional bacterial group after prolonged storage. Besides, the abundance of methane-oxidizing bacteria was significantly increased in synergy with PBB and denitrifiers to achieve reactivating and stable operation of anaerobic methane oxidation coupled to denitrification.Graphical abstract: