Two parallel membrane bioreactors (MBRs) were operated under different calcium dosages (168.5, 27 mg/L) to gain a better understanding of the mechanism of retarding membrane fouling by adding calcium. The results showed that the particle size of sludge flocs increased and the particle size distribution tended to be narrow at the optimum dosage (168.5 mg/L). Calcium was effective in decreasing loosely bound extracellular polymeric substances (LB-EPS) in microbial flocs and soluble microbial products (SMP) in the supernatant at the dosage of 168.5 mg/L by strengthening the neutralization and bridging of EPS with flocs. Furthermore, the amount of CODs and CODc decreased in both the mixed liquor and the fouling cake layer on the membrane surface. In order to compare the filtration characteristics of cake layers from the MBRs with the two calcium dosages, the specific cake resistance and the compressibility coefficient were measured. The specific cake resistance from the MBR with optimum dosage (168.5 mg/L) was distinctly lower than that with low dosage (27 mg/L). The compressibility coefficient of the cake layers under two dosages were respectively attained as 0.65, 0.91. Scanning electron microscopy (SEM) and three-dimensional confocal scanning laser microscope analysis (CLSM) images were utilized to observe the gel layer directly.