We evaluated uranium bioleaching from low-grade, granite-type uranium ore using mixed acidophilic microbes from uranium mine leachate. A 4854-ton plant-scale heap bioleaching process achieved sustained leaching with a uranium leaching efficiency of 88.3% using a pH of 1.0–2.0 and an Fe3+ dosage of 3.0–5.5 g/L. Acid consumption amounted to 25.8 g H2SO4 kg−1 ore. Uranium bioleaching follows a diffusion-controlled kinetic model with a correlation coefficient of 0.9136. Almost all uranium was dissolved in aqueous solution, except those encapsulated in quartz particles. Therefore, heap bioleaching by mixed acidophilic microbes enables efficient, economical, large-scale recovery of uranium from low-grade ores.