Excess sulfate production and low concentration of perchlorate removal are the main problems for sulfur-based perchlorate reduction reactor. In this study, the problems were firstly solved by step-feeding under mixotrophic conditions. The performances of step-feed sulfur-based reactor SFSBR) and up-flow sulfur-based reactor UFSBR) are compared. At perchlorate of 194 mg/L, acetate of 28.8 mg/L and hydraulic retention time of 0.9 h, the Half-order reaction rate constant and the sulfate production of SFSBR were 29.7 mg(1/2)/L-1/2.h and 171 mg/L, respectively, which were superior to those of UFSBR. The oxidation-reduction potential values of SFSBR were lower than that of UFSBR. Meanwhile, the biodiversity along the height of the reactor was decreased by step-feeding. Principal component analysis showed significant interrelations existed among the bacterial community composition and the operational/environmental conditions in each treatment zone. Consequently, the SFSBR provides an effectively alteration for the removal of high perchlorate concentration and control sulfate.