Vanadium is a commercially important metal that is released into the environment by fossil fuel combustion and mining. Despite its prevalence as a contaminant, the potential for vanadium bioremediation has not been widely studied. Injection of acetate (as a carbon source) directly into an aquifer to biostimulate contaminated sediments in Colorado, United States, resulted in prolonged removal of aqueous vanadium for a period of at least two years. To further investigate this process, we simultaneously added acetate and vanadate (V(5+)) to columns that were packed with aquifer sediment and inserted into groundwater wells installed on the Colorado River floodplain. This allowed evaluation of the microbial response to amendments in columns that received an influx of natural groundwater. Our results demonstrate the removal of up to 99% of the added V(5+)(aq) and suggest microbial mediation. Most probable number measurements demonstrate up to a 50-fold increase in numbers of V(5+)-reducing cells in vanadium-amended columns compared to controls. 16S rRNA gene sequencing indicates decreased diversity and selection for specific taxa in columns that received vanadate compared to those that did not. Overall, our results demonstrate that acetate amendment can be an effective strategy for V removal, and that V bioremediation may be a viable technology.