Microbial vanadate (V(V)) reduction is a key process for environmental geochemistry and detoxification of vanadium (V). However, the electron transfer pathways and V isotope fractionation involved in this process are not yet fully understood. In this study, the V(V) reduction mechanisms with concomitant V isotope fractionation by the Gram-positive bacterium Bacillus subtilis (B. subtilis) and the Gram-negative bacterium Thauera humireducens (T. humireducens) were investigated. Both strains could effectively reduce V(V), removing (90.5% ± 1.6%) and (93.0% ± 1.8%) of V(V) respectively from an initial concentration of 50 mg·L⁻¹ during a 10-day incubation period. V(V) was bioreduced to insoluble vanadium (IV), which was distributed both inside and outside the cells. Electron transfer via cytochrome C, nicotinamide adenine dinucleotide, and glutathione played critical roles in V(V) reduction. Metabolomic analysis showed that differentially enriched metabolites (quinone, biotin, and riboflavin) mediated electron transfer in both strains. The aqueous V in the remaining solution became isotopically heavier as V(V) bioreduction proceeded. The obtained V isotope composition dynamics followed a Rayleigh fractionation model, and the isotope enrichment factor (ε) was (–0.54‰ ± 0.04‰) for B. subtilis and (–0.32‰ ± 0.03‰) for T. humireducens, with an insignificant difference. This study provides molecular insights into electron transfer for V(V) bioreduction and reveals V isotope fractionation during this bioprocess, which is helpful for understanding V biogeochemistry and developing novel strategies for V remediation.