Abstract: To elucidate the operational regulation mechanisms of enhanced biological phosphorus removal systems under low-carbon conditions, an enhanced biological phosphorus removal system dominated by Tetrasphaera and Candidatus Accumulibacter was established under low-carbon conditions using casein hydrolysate as the sole carbon source. Furthermore, the effects of operational mode transition on the removal performance of various pollutants were investigated by shortening the anaerobic phase from 4 h to 2 h and introducing an additional 2 h anoxic phase. After 100 days, the system’s overall performance reached full stability; by the end of the first stage, the removal efficiencies of PO₄³⁻-P and NH₄⁺-N were both above 85%, and the COD removal efficiency reached approximately 90%. After the operational mode shift, phosphorus removal efficiency decreased slightly but still remained above 80%. In addition, the variations in pollutant removal performance were highly consistent with the succession patterns of the microbial community. After 120 days of enrichment and acclimation, the relative abundances of Tetrasphaera and Candidatus Accumulibacter increased to 2.45% and 3.79%, respectively, while the proportion of the main nitrifying genus Nitrospira rose from 0.02% to 2.79%. Under the A²/O operational mode, the relative abundances of various denitrifying genera increased significantly, with Thauera reaching 12.93%. Due to the shortened anaerobic duration and reduced energy utilization efficiency, the growth of polyphosphate-accumulating organisms was affected, with the relative abundances of Tetrasphaera and Candidatus Accumulibacter declining to 2.03% and 3.03%, respectively. Meanwhile, the denitrifying polyphosphate-accumulating genus OLB12 exhibited strong adaptability to the anoxic environment, with its relative abundance further increasing to 4.79%. In summary, under low-carbon conditions, the microbial community is relatively sensitive to changes in operational mode, and with appropriate carbon sources and phase duration configuration, the enhanced biological phosphorus removal system shows potential for simultaneous nitrogen and phosphorus removal.