Nitrate-dependent anaerobic methane oxidation (N-DAMO), a bioprocess that couples the oxidation of green gas and the removal of nitrogen oxides in a microbial group, has gained much attention as a potential economical method of biological removal of nitrates and methane from wastewater. Low-temperature (20 °C) operation of N-DAMO would be beneficial to utilize the methane dissolved in the effluent and thus decrease the cost of maintaining the bioreactor temperature in wastewater treatment. Here, the long-term (>350 days) operational activities of N-DAMO were evaluated to assess the performance of N-DAMO from stepwise cooling (30-20 °C) to ambient temperatures (13-38 °C). Under stepwise cooling conditions, the activity of the N-DAMO community was first inhibited and then rapidly adjusted. Notably, a similar N-DAMO activity was observed at 30 °C and 20 °C. Under ambient temperature conditions, the highest nitrate removal rate observed at the beginning of the test was 7.14 mg-N/L/d, which was 5.3 times higher than that recorded at the end of the test. This indicates that the long-term temperature fluctuation irreversibly inhibited N-DAMO activity. 16S rRNA gene sequencing analyses found that the functional archaea were ANME-2D, which has been deemed as the dominant culture in the N-DAMO process. The abundance of ANME-2D on the last day at stepwise cooling temperature conditions was much higher than on day 0, but disappeared after a long period of operation at ambient temperature. It was assumed that N-DAMO would stabilize at stepwise cooling temperature conditions, but not at ambient temperature. Our findings could offer a promising technology for anaerobic wastewater treatment plants (WWTPs) in temperate and warm climate zones.