Calcium-rich aerobic granules were cultivated after 3-month operation. The chemical form and spatial distribution of calcium in the granules and their physicochemical characteristics were explored. Examination with a scanning electron microscope combined energy dispersive X-ray detector (SEM–EDX) shows that Ca was mainly accumulated in the core of the granules. CaCO 3 was found to be the main calcium precipitate in the granules. The fluorescent in situ hybridization (FISH) analysis shows that the cells were crowded in the outer layer and gathered in clusters. Compared with the granules without Ca accumulation, the Ca-rich granules had more rigid structure and a higher strength. However, their specific oxygen uptake rate (SOUR) reduced after the Ca accumulation inside them. Comparison between the SOUR values of the granules with and without Ca accumulation suggests that Ca accumulated in the aerobic granules might have a negative effect on their bioactivity.