The ex-situ bioremoval of U(VI) from contaminated water using Acidithiobacillus ferrooxidans strain 8455 and 13538 was studied under a range of pH and uranium concentrations. The effect of pH on the growth of bacteria was evaluated across the range 1.5–4.5 pH units. The respiration rate of At. ferrooxidans at different U(VI) concentrations was quantified as a measure of the rate of metabolic activity over time using an oxygen electrode. The biosorption process was quantified using a uranyl nitrate solution, U-spiked growth media, and U-contaminated mine water. The results showed that both strains of At. ferrooxidans are able to remove U(VI) from solution at pH 2.5–4.5, exhibiting a buffering capacity at pH 3.5. The respiration rate of the micro-organism was affected at U(VI) concentration of 30 mg L−1. The kinetics of the sorption fitted a pseudo-first order equation, and depended on the concentration of U(VI). The KD obtained from the biosorption experiments indicated that strain 8455 is more efficient for the removal of U(VI). A bioreactor designed to treat a solution of 100 mg U(VI) L−1 removed at least 50% of the U(VI) in water. The study demonstrated that At. ferrooxidans can be used for the ex-situ bioremediation of U(VI) contaminated mine water