In this study, the manganese (Mn) reduction-coupled denitrification strategy of dissimilatory Mn reducing bacteria was insightfully investigated. Different parameters (MnO2 level, pH, and temperature) were optimized by kinetic fitting to improve denitrification and Mn reduction effects. The 300 mg L−1 MnO2 addition achieved 98.72% NO3–-N removal in 12 h, which was 54.62% higher than blank group without MnO2. Scale-up studies showed that the metabolic activity of the bacteria was effectively enhanced by the addition of MnO2. Besides the deepening of humification in the system, tryptophan-like protein and polysaccharide as potential electron donor precursors revealed remarkable contributions to the extracellular secretion-dependent denitrification process of DMRB. The effect of EPS on Mn reduction depends mainly on the capture of MnO2 by the LB-EPS layer versus its dissolution in the TB-EPS layer. Ultimately, the EPS possess a dual effect of accelerated denitrification and Mn reduction efficiency due to the enhanced EET process.