In this study, the effect of three Mg/Ca molar ratios (5.0, 3.8 and 1.7) on denitrifying phosphate removal performance, biomass morphology, and Extracellular Polymeric Substances (EPS) were examined. Results showed that when the influent Mg/Ca molar ratio was 3.8, the anaerobic-anoxic EBPR performed complete phosphate removal. The microbial bacterial population was a mixed culture comprised of 81 ± 3% DPAO and 13 ± 2% denitrifying glycogen accumulating organisms (DGAO). A higher influent Mg/Ca molar ratio (5.0) had a distinct impact on phosphate removal, biomass morphology, and EPS. This probably induced the deterioration of the anaerobic-anoxic Enhanced Biological Phosphorus Removal (EBPR). The results of this study may inform the proper operation of an anaerobic-anoxic EBPR, and contribute to its application in the real world.