Partial denitrification coupled with anammox (PD/A) is a promising energy-efficient alternative for mainstream nitrogen removal, yet its stability under different reactor modes remains unclear. This study systematically compared sequencing batch (RS) and continuous-flow (RC) PD/A systems using performance monitoring, activity assays, and metagenomic analyses. RC maintained superior long-term stability, achieving >80% nitrogen removal with anammox contributing above 90%, whereas RS remained less stable (∼65% NRE, ∼75% anammox contribution) and relied more on complete denitrification. Activity assays revealed that PD activity in RS was 5.03 times that of anammox, favoring complete denitrification. In contrast, RC maintained balanced activities, with PD activity being 0.89 times that of anammox, supporting cooperative PD/A interactions. Metagenomic analysis further revealed that Thauera-associated Nir/Nor/Nos genes were markedly enriched in RS, indicating a stronger potential for complete denitrification. These findings demonstrate that reactor mode regulates PD/A interactions through distinct stabilization mechanisms, with continuous-flow operation enhancing microbial cooperation and system resilience. This study provides mechanistic insights and practical guidance for the stable implementation of mainstream PD/A processes.