Stored Anammox sludge (SAS) was used in an expanded granular sludge bed (EGSB) reactor treating synthetic wastewater with the aim of evaluating its possible use as seed sludge. The SAS had been kept in a refrigerator (4 °C) without any feed. After 2 years, only 1–2% Anammox bacteria could survive in the SAS. However, it soon prevailed in the EGSB reactor after loading. Accordingly, the start-up of the EGSB reactor was successfully completed in 34 days. The biomass turned to round reddish granular sludge from irregular brown floc at the end of this study. The results indicate that SAS could serve well as seed sludge. The required time for start-up of the Anammox reactor using SAS was thus demonstrated to be shorter than that of uncultivated sludge under experimental conditions.