This study investigated the effects of varying aerobic/hypoxia alternation number on organic removal performance in a sequencing batch reactor (SBR) and elucidated the underlying mechanisms through nitrogen transformation rates, enzyme activities, microbial community structure, and functional gene abundance. As the alternation frequency increased from 1 to 4, nitrite accumulation in the effluent gradually intensified, accompanied by declines in nitrite oxidation rates, nitrate reduction rates, and significant reductions in nitrite oxidoreductase and nitrate reductase activities. Metagenomic analysis revealed that increased alternation frequencies universally reduced the relative abundances of nitrite-oxidizing bacteria (e. g., Nitrospira) and denitrifiers (e. g., Hyphomicrobium and Defluviicoccus), while diminishing the abundances of nitrogen-transforming enzymes and their encoding functional genes (e. g., nxrAB, nirK, and nirS). These findings collectively explain the mechanisms of nitrite accumulation and impaired nitrogen transformation efficiency from microbial, enzymatic, and genetic perspectives. This work provides theoretical and technical insights for evaluating the impacts of aerobic/hypoxia alternation number on nitrogen removal performance, microbial community dynamics, and functional gene regulation in SBR systems.