The short-term and long-term effects of alanine on ANAMMOX process were investigated by batch experiments. The ANAMMOX process was greatly affected both in short-term and long-term culture when alanine was the sole substrate, and there was no nitrogen removal in the system. Without electron acceptor NO₂^--N, ANAMMOX process could not occur in the system, although the alanine removal could reach more than 86%, NH₄⁺-N was accumulated in the system. When NH₄⁺-N and NO₂^--N were added to the system with alanine as a substrate, the activity of ANAMMOX bacteria was not affected so much in the short term (7h), and 2mmol/L and 10mmol/L alanine reached 78% and 99% removal efficiencies in 10 and 60h, respectively. During the long term experiments, the activity of ANAMMOX bacteria could be inhibited by alanine with a concentration of 2mmol/L. Combined with ANAMMOX and denitrification processes, the removal efficiency of TN reached 57%, and the removal efficiency of alanine was about 99%.