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The preservation of anammox granules is of great significance for the rapid start-up of the anammox process and improvement of performance stability. Therefore, it is necessary to explore an economical and stable preservation strategy. Exogenous extracellular polymeric substances (EPS) were used as protective agents for the preservation of anammox granules in this study. In brief, EPS from anammox sludge (A-EPS) and denitrifying sludge (D-EPS) were added to preserve anammox sludge at 4 °C and room temperature (15–20 °C). The results showed that A-EPS addition at 4 °C was the optimal condition for the preservation of anammox granules. After 90 days of preservation, the specific anammox activity (SAA) of the anammox granules remained at 92.7 ± 2.2 mg N g−1 VSS day−1 (remaining ratio of 33.4%), while that of the sludge with D-EPS addition at the same temperature was only 77.1 ± 3.2 mg N g−1 VSS day−1 (remaining ratio of 27.8%). The nitrogen removal efficiency of the experimental group with D-EPS at room temperature was 85.9%, and that of the A-EPS group reached 90.6% under the same temperature conditions. The abundance of the functional genes hzs A, hdh and nir S of the sludge (4 °C; A-EPS addition) after recovery were 138.5%, 317.1%, and 375.9%, respectively, of those of sludge from the D-EPS-added group at the same temperature. RDA revealed the contribution of proteins to the preservation process. Overall, this study provides an economical and robust strategy for the preservation of anammox granules. Unlabelled Image • Exogenous EPS was used as protective agents for the anammox sludge preservation. • Addition of exogenous EPS significantly increased anammox activity in short term. • Adding A-EPS at 4 °C led to the optimal recovery performance. • The protein in EPS is a potential factor in optimizing preservation performance. [ABSTRACT FROM AUTHOR]