The sludge settleability is of prime importance for the activated sludge process. The effect of calcium ion on the biological performance of sludge was investigated in a lab-scale activated sludge system with varying Ca2+ concentration. Results indicated that addition of 150 mg/L Ca2+ to the influent significantly improved the settling characteristics and metabolic reactivity of activated sludge in the bioreactors. Analyses using denaturing gradient gel electrophoresis (DGGE) and 16S rRNA sequencing showed that a significant difference in the presence of certain bacterial groups between the sludge systems with 150 mg/L Ca2+ and those with 0-100 mg/L Ca2+ addition. Ca2+ also increased the production of the extracellular polymeric substance (EPS) and facilitated the development of microbial cluster in the bioreactor. Study showed that an addition of 150 mg/L Ca2+ to the influent provides a simple approach to improve the settling properties of activated sludge and maintain high pollutant removal efficiency. (C) 2016 Elsevier Ltd. All rights reserved.