The problem of co-occurring nitrate and vanadium contamination is posing a serious risk to groundwater ecosystem. Although heterotrophic microbial remediation strategies for NO3−-N or V(V) have been reported, little is known regarding the selection of PRB materials and environmental risk assessment during co-contamination remediation. Herein, this study compared the pollutant removal load and removal performance of four fillers over 120-d to screen for the best fillers (CB: nitrate (36.85 mg/g), V(V) (2.07 mg/g)). UV–vis and fluorescence spectra were used to analyze the distribution of dissolved organic matters (DOM) in effluent and to assess the possible environmental risk. The distribution of microbial community structure revealed that the type of carbon source was one of the important factors to shape the community structure. Based on metagenomic techniques, the advantage of co-pollution removal in the CB system were energy storage and preferential electron utilization of NH4+-N and organic nitrogen production. Moreover, the detoxification of vanadium in CB was mainly mediated by receiving electrons from the electron transfer chain, while vanadium in GL was mainly mediated by NarG and NapA. The phenomenon of V(V) re-release during the experiment completes our understanding of the biogeochemical cycling of vanadium, nitrogen and carbon. Overall, the findings of this study provide new insights into remediation strategies for NO3−-N and V(V) co-pollutants, and can provide guiding recommendations for the selection and optimization of technologies and fillers in the remediation of co-pollutants in actual sites.