Antimony contamination from textile industries has been a global environmental concern and the existing treatment technologies could not reduce Sb(V) to meet the discharge standards. To overcome this shortcoming, ferric flocs were introduced to expedite the biological process for enhanced Sb(V) removal in wastewater treatment plant (WWTP). For this purpose, a series of laboratorial-scale sequential batch reactor activated sludge processes (SBRs) were applied for Sb(V) removal with varied reactor conditions and the transformation of Fe and Sb in SBR system was investigated. Results showed a significant improvement in Sb(V) removal and the 20 mg L−1 d−1 iron ions dosage and iron loss rate was found to be only 15.2%. The influent Sb(V) concentration ranging 153–612 μg L−1 was reduced to below 50 μg L−1, and the maximum Sb(V) removal rate of the enhanced system reached about 94.3%. Furthermore, it exhibited high stability of Sb(V) removal in the face of antimonate load, Fe strike and matrix change of wastewater. Sludge total Sb determination and capacity calculation revealed decreasing in Sb adsorption capacity and desorption without fresh Fe dosage. While sludge morphology analysis demonstrated the aging and crystallization of iron hydroxides. These results verify the distinct effects of fresh iron addition and iron aging on Sb(V) removal. High-throughput gene pyrosequencing results showed that the iron addition changed microbial mechanisms and effect Fe oxidized bacterial quantity, indicating Sb(V) immobilization achieved by microbial synergistic iron oxidation. The present study successfully established a simple and efficient method for Sb(V) removal during biological treatment, and the modification of biological process by iron supplement could provide insights for real textile wastewater treatment.