High calcium content has been widely reported in acetate-fed aerobic granules, but the reason behind this is unclear yet. By SEM-energy dispersive X-ray mapping analysis, this study showed that the majority of calcium was presented in the central part of the acetate-fed aerobic granule, and the granule shell part was nearly calcium-free. The elemental analysis of calcium ions coupled with the chemical titration of carbonate further revealed that the calcium ions that accumulated in the acetate-fed aerobic granule mainly existed in the form of calcium carbonate (CaCO3). The formation of the CaCO3 appeared to be highly dependent on the size of the aerobic granule, i.e., the CaCO3 precipitation was found only in aerobic granules with radiuses larger than 0.5 mm. These experimental observations with regard to the formation of CaCO3 in the acetate-fed aerobic granule were further confirmed by the model simulation, which was based on the principles of mass diffusion and carbonate dissociation in liquid phase. This study for the first time showed that the size of the acetate-fed aerobic granule would indeed play an essential role in the CaCO3 formation, and provided experimental evidence that a crystal CaCO3 core was not necessarily required for granulation.