In this study, a novel high-frequency micro-aeration/anoxic (HMOA) mode with a high aeration frequency (15 times/h) and short aeration duration (Taer = 1 h/cycle) was proposed. Compared with continuous aeration modes, the highest nitrogen and phosphorus removal efficiencies were achieved in the sequencing batch reactor (SBR) under HMOA mode when treating wastewater with carbon/nitrogen (C/N) ratios of 4.5 (85% and 97%, respectively) and 3 (77% and 75%, respectively). Metagenomic analysis was utilized to analyse the microbial metabolic mechanism under the HMOA mode. The results showed that under the HMOA mode, the enhanced transduction and metabolism pathways of nitrate, nitrite, oxygen, phosphorus and acetate provided favourable nutritional conditions for the proliferation of denitrifiers and phosphorus accumulating organisms (PAOs), and simultaneously strengthened the survival capacity of nitrifiers under low dissolved oxygen (DO) conditions. In addition, genes involved in carbon metabolism were upregulated by the HMOA mode, which further increased the utility of carbon sources for denitrifier and PAO metabolism. Consequently, the limited carbon source could be fully utilized in nitrogen and phosphorus removal, which improved the efficiency of treating low C/N wastewater. This study proposed a potential aeration mode for microbial metabolism regulation to enhance nutrient removal in biological wastewater treatment processes.