Denitrification in mixed culture system has been extensively researched to date, but few studies have focused on accelerating the process using redox mediators to promote electron transfer. Strain L2, an iron-reducing bacteria, can remove 75.44% of nitrate under temperature of 30.60 °C, pH of 6.75 and Fe2+ concentration of 27.86 mg·L-1. Additionally, the removal rate of nitrate reached 1.516 mg·L-1·h-1 in 8 h with the addition of 0.030 mmol·L-1 2-hydroxy-1,4-naphthoquinone (HNQ), which increased by 1.38 times than control group. Furthermore, analysis by fluorescence spectroscopy, flow cytometer and gas chromatography demonstrated that HNQ positively stimulated denitrification. This study provides a reference for enhancing denitrification in mixed culture and lays the foundation for the practical application of redox mediators in groundwater treatment.