A direct step-wise regulation strategy of microbial community structure was developed for improving chalcopyrite bioleaching by Acidithiobacillus sp. Specially, the initial microbial proportion between Acidithiobacillus ferrooxidans and Acidithiobacillus thiooxidans was controlled at 3:1 with additional 2 g/L Fe2+ for faster initiating iron metabolism. A. thiooxidans biomass was fed via a step-wise strategy (8-12th d) with the microbial proportion 1:1 for balancing community structure and promoting sulfur metabolism in the stationary phase. A. thiooxidans proportion was further improved via another step-wise feeding strategy (14-18th d) with the microbial proportion 1:2 for enhancing sulfur metabolism and weakening jarosite passivation in the later phase. With the community structure-shift control strategy, biochemical reaction was directly regulated for creating a better balance in different phases. Moreover, the final copper ion was increased from 57.1 to 93.2 mg/L, with the productivity 2.33 mg/(L d). The novel strategy may be valuable in optimization of similar bioleaching process. (C) 2015 Elsevier Ltd. All rights reserved.