The decreased activity (i.e. decay) of anaerobic ammonium oxidation (Anammox) bacteria during starvation can be attributed to death (i.e. decrease in the amount of viable bacteria) and activity decay (i.e. decrease in the specific activity of viable bacteria). Although they are crucial for the operation of the Anammox process, they have never been comprehensively investigated. This study for the first time experimentally assessed death and activity decay of the Anammox bacteria during 84 days’ starvation stress based on ammonium removal rate, Live/Dead staining and fluorescence in-situ hybridization. The anaerobic and aerobic decay rates of Anammox bacteria were determined as 0.015 ± 0.001 d⁻¹ and 0.028 ± 0.001 d⁻¹, respectively, indicating Anammox bacteria would lose their activity more quickly in the aerobic starvation than in the anaerobic starvation. The anaerobic and aerobic death rates of Anammox bacteria were measured at 0.011 ± 0.001 d⁻¹ and 0.025 ± 0.001 d⁻¹, respectively, while their anaerobic and aerobic activity decay rates were determined at 0.004 ± 0.001 d⁻¹ and 0.003 ± 0.001 d⁻¹, respectively. Further analysis revealed that death accounted for 73 ± 4% and 89 ± 5% of the decreased activity of Anammox bacteria during anaerobic and aerobic starvations, and activity decay was only responsible for 27 ± 4% and 11 ± 5% of the decreased Anammox activity, respectively, over the same starvation periods. These deeply shed light on the response of Anammox bacteria to the starvation stress, which would facilitate operation and optimization of the Anammox process.