Although aerobic granulation has been intensively studied, the possible mechanism of this cell-to-cell self-immobilization phenomenon still remains unclear. Aerobic granulation in the absence and presence of a chemical uncoupler, 3,3',4',5-tetrachlorosalicylanilide (TCS), which can dissipate the proton gradient and further disrupt ATP synthesis, was investigated. Upon exposure to TCS, precultivated mature aerobic granules underwent disintegration, indicating that the stability and integrity of aerobic granules would be associated with microbial energy metabolism. It was also shown that the formation of aerobic granules in the presence of TCS was completely inhibited as compared with the control free of TCS. These results, for the first time, reveal that aerobic granulation is energy metabolism dependent, and possible reasons are also discussed.