<p>The co-occurrence of <i>N,N</i>-dimethylformamide (DMF) and residual nitrogen in industrial wastewater presents significant challenges for conventional biological treatment. To overcome these limitations, this study developed an innovative zero-valent iron (ZVI) enhanced nitrate-reducing bioreactor (ZVI-NR) that establishes an efficient electron transfer pathway for simultaneous DMF mineralization and nitrate removal. The ZVI-NR system achieved complete DMF removal (100%) and high nitrate reduction efficiency (96.17% ± 1.50%), representing 40.37% ± 2.30% and 34.23% ± 1.30% improvements over conventional NR system. The key innovation involves establishing an Fe²⁺/Fe³⁺ electron shuttle system, coupled with the selective enrichment of iron-cycling genera (such as <i>Dojkabacteria</i> and <i>Denitratisoma</i>). These genera maintain iron bioavailability and facilitate extracellular electron transfer. The increased enzymatic activity (136.96%–161.23% for nitrate/nitrite reductases), and dynamic extracellular polymeric substance (EPS) secretion (154.73 ± 4.65 mg/g VSS) featuring α-helix-dominated protein structures that improve microbial aggregation (<i>Dojkabacteria, Chryseobacterium</i> and <i>Arenimonas</i>, etc.). The superior performance of the system is further attributed to dual metabolic regulation through <i>feoAB</i>-mediated Fe²⁺ transport and a formate dehydrogenase mediated mechanism that is hypothesized to contribute to the proton motive force. This study demonstrates a technological breakthrough in industrial wastewater treatment, which achieves stable and complete DMF mineralization at high loading rates. The unique iron-mediated electron transfer that enhances efficient DMF degradation and optimized nitrogen removal, addressing the challenge of treating refractory wastewater containing high organic and nitrogen loads.</p>