Iron-oxidizing bacteria played an important role in the treatment of Sb-containing wastewater. In this study, effect of different iron sources on Sb(III) removal ability by isolated iron-oxidizing bacteria (named as IOB-L) was conducted systemically in batch experiment. Moreover, ferrous lactate and zero-valent iron were chosen as iron sources for IOB-L. The results showed that after inoculation of 2% volume of IOB-L, Sb(III) concentration in water decreased from initial 18 mg/L to 4.1 mg/L at optimal pH of 7.0. There was no reaction between Sb(III) and ferrous lactate, whereas corrosion product of iron can adsorb certain amount of Sb. When active IOB-L cultivated in ferrous lactate, a better removal rate of Sb(III) can be reached with a longer stagnate phase for bacteria. However, Sb(III) removal ability of IOB-L using zero-valent iron as iron source was lower. SEM-EDS, FTIR, and XPS analysis further indicated that ferrous lactate was oxidized by IOB-L and precipitated as biogenic iron oxides which had strong adsorption ability towards Sb(III), whereas zero-valent iron was not a good iron source.