Schwertmannite biosynthesized by resting Acidithiobacillus ferrooxidans (A.ferrooxidans) with FeSO4 has high purity and specific surface area. It plays an important role in the removal of toxic heavy metals or metalloid in water environmental. To provide the optimum parameters for schwertmannite scale production, the effects of storage time of resting A.ferrooxidans cells and reuse of the recycling cells on the strain growth and its ability in facilitating Fe2+ oxidation and mineral formation were investigated through the flask experiments. The results indicated that the fresh resting A.ferrooxidans cells harvested within 30 d could completely oxidize 144 mmol L-1 Fe2+ to Fe3+ after 48 h in the system with initial pH 2.50. In the presence of excess SO42-, about 41.99% of the ferrous iron was transformed into the brown-red schwertmannite, and mineral weight reached 1.34 g in 250 mL system. Compared to fresh resting A.ferrooxidans cells, the Fe2+ oxidation percentage by the relatively aged resting A.ferrooxidans cells stored for 45 d at 4 °C was decreased by 5.61%, and the schwertmannite weight was decreased by 35.07%. Furthermore, the reuse of resting A.ferrooxidans cells resulted in a significant reduction of Fe2+ oxidation with only 1/7 of the Fe2+ oxidation ability of fresh resting cells, which was contributed to the oxidation ability decline of the reused cells itself and the cell density decrease of A.ferrooxidans due to absorption or wrapping by minerals. Therefore, the newly-prepared resting A.ferrooxidans cells should be used in time or its storage time should not exceed 30 d in the acidic solution at 4 °C. The resting A.ferrooxidans cells could be recycled and used for the biosynthesis of schwertmannite, but its efficiency was only about 15% of newly-prepared resting cells.