Whereas prospects of bioremediation for a vanadium(V) [V(V)]-contaminated environment are widely recognized, reported functional species are extremely limited, with the vast majority of Gram-negative bacteria in Proteobacteria. Herein, the effectiveness of V(V) reduction is proved for the first time by Lactococcus raffinolactis, a Gram-positive bacterium in Firmicutes. The V(V) removal efficiency was 86.5 ± 2.17% during 10-d operation, with an average removal rate of 4.32 ± 0.28 mg/L·d in a citrate-fed system correspondingly. V(V) was bio-reduced to insoluble vanadium(IV) and distributed both inside and outside the cells. Nitrite reductase encoded by gene nirS mainly catalyzed intracellular V(V) reduction, revealing a previously unrecognized pathway. Oxidative stress induced by reactive oxygen species from dissimilatory V(V) reduction was alleviated through strengthened superoxide dismutase and catalase activities. Extracellular polymeric substances with chemically reactive hydroxyl (-OH) and carboxyl (-COO-) groups also contributed to V(V) binding and reduction as well as ROS scavenging. This study can improve the understanding of Gram-positive bacteria for V(V) bio-detoxification and offer microbial resources for bioremediation of a V(V)-polluted environment.