Bioleaching Xiangjiang River alkaline sediment contaminated by multiple heavy metals was investigated. Multiple metals in alkaline sediment possess significant toxicity to aquatic organisms or humans and will greatly inhibit bioleaching. The bioleaching method using autotrophic bacteria mixed with heterotrophic bacteria can solve this problem successfully. The experiment results showed that bioleaching efficiencies of Zn, Mn, Cu, and Cd were 95.2 %, 94.2 %, 90.1 %, and 84.4 %, respectively. Moreover, the changes of heavy metal concentrations in different fractions in contaminated sediment before and after bioleaching were analyzed by selective sequential extraction, and it was discovered that the main fractions of Zn, Mn, Cu and Cd after bioleaching are Fe-Mn oxide, organic associated form and a residual form. Its biotoxicity decreased greatly. The bioleaching heavy metals from sediment using autotrophic bacteria combined with heterotrophic bacteria can effectively improve the bioleaching efficiency and reduce toxicity.