Vanadium (V) is a common heavy metal and often co-occurs with nitrate in effluents from mining and metal finishing industry. In the present study, the toxic effects of V(V) were examined in a sulfur and hydrogen based autotrophic denitrification system. This combined system achieved simultaneously microbial denitrification and V(V) reduction. High concentration of V(V) (60 and 100 mg/L) inhibited the denitrification activities, while 30 mg/L V(V) had a very slight effect. V(V) induced increases of lactate dehydrogenase release and reactive oxygen species production, which may inhibit nitrate and nitrite reductases activities and abundances of denitrifying functional genes. Moreover, the extracellular polymeric substance production was also suppressed under V(V) stress, thereby decreasing the amount of biofilm biomass. Microbial community analyses suggesting the genus Bacillus may have higher tolerance to V(V). These findings can provide scientific basis for the optimized design of treatment system to remove nitrate and V(V) simultaneously.