Abstract Toxic ions accumulation in leachate is one of the limitations for bioleaching application. The effects of fluoride ions on microbial growth relevant to bioleaching have been extensively emphasized. However, the reasons that the microorganisms present different fluoride tolerance in bioleaching of different minerals have not yet been proposed. A fundamental exploration on the differential fluoride tolerance of sulfur- and ferrous iron-grown Acidithiobacillus ferrooxidans was performed. The results showed that the critical fluoride concentration for sulfur-grown A. ferrooxidans was less than 0.1 mM whenever fluoride was introduced. The critical fluoride concentration for ferrous iron-grown A. ferrooxidans was 1.0 mM when fluoride was introduced prior to the lag phase, but it was higher than 10.0 mM when fluoride was introduced at the log phase. The ferric ions generated from ferrous oxidation could significantly decrease the concentration of HF and finally reduce the toxicity of fluoride to A. ferrooxidans. The changes of concentration of metal ions with strong complex ability to fluoride ions, resulted in an illusion of high fluorine-resistant strain in industrial bioleaching heap. Finally, the feasibility of fluoride removal by ferric ion was put forward. This work has meaningful implications for the industrial bioleaching of high-fluorine containing uranium ore.