Water hardness is a critical yet often overlooked parameter in enhanced biological phosphorus removal (EBPR). While divalent cations are known to influence chemical phosphorus removal, their impact on biological processes—especially microbial phosphorus transformation and feedback mechanisms—remains unclear. Therefore, four anaerobic/aerobic/anoxic(A/O/A) sequencing batch reactor (SBR) with different hardness levels (0, 150, 300, and 600 mg/L as CaCO₃) were constructed and operated in this study. Results demonstrated enhanced phosphorus removal under high hardness, due to two synergistic effects: divalent cations combined with anaerobically released phosphate to form inorganic minerals, alleviating phosphate accumulating organisms (PAOs) inhibition and improving floc stability; and hardness increased key enzyme (PPK and PPX) activities, boosting PAOs phosphorus metabolism. Phosphorus speciation shifted from soluble and weakly-bound to stable Ca/Mg-bound and residual forms, with ³¹P MAS NMR confirming elevated orthophosphate, polyphosphate, and inorganic phosphorus signals under higher hardness. These findings illustrate a synergistic “biological phosphorus uptake + chemical precipitation” mechanism. Microbial analysis showed high hardness suppressed glycogen-accumulating organisms (GAOs), raising the PAOs/GAOs ratio. Genetically, hardness upregulated the global regulatory gene phoR, coordinating phosphorus uptake, storage, and transformation. In summary, increased hardness enhanced EBPR efficiency through phoR-mediated regulation and biochemical synergy, offering a theoretical and strategic basis for EBPR in high-hardness water regions.