Polymetallic mine tailings have great potential as carbon sequestration tools to stabilize atmospheric CO 2 concentrations. However, previous studies focused on carbonate mineral precipitation, while the role of autotrophs in carbon sequestration by mine tailings has been neglected. In this study, carbon sequestration in two mine tailings treated with FeS 2 and 13 C-labeled CO 2 was analyzed using 13 C isotope labeling, pyrosequencing and DNA-based stable isotope probing (SIP) to identify carbon fixers. Mine tailings treated with FeS 2 exhibited a higher percentage of 13 C atoms (1.76 ± 0.06 in Yangshanchong and 1.36 ± 0.01 in Shuimuchong) than the control groups over a 14-day incubation, as well an increase in the total organic carbon (TOC) content (0.20 ± 0.11 mg/g in Yangshanchong and 0.28 ± 0.14 mg/g in Shuimuchong). These data demonstrated the role of autotrophs in carbon sequestration with pyrite addition. Pyrite treatment led to changes in the composition of bacterial communities, and the genera Sulfobacillus (8.04 %) and Novosphingobium (8.60 %) were found to be dominant in these communities. In addition, the DNA-SIP results indicated that the cbbL gene copy number was 8.20–16.50 times greater than the cbbL gene copy number in 13 C-labeled heavy fractions. Furthermore, a Sulfobacillus -like cbbL gene sequence (cbbL-OTU1) accounted for 30.11–34.74 % of all cbbL gene sequences in the 13 C-labeled heavy fractions of mine tailings treated with FeS 2 . These findings highlight the importance of the RubisCO form I-encoding gene, cbbL , in bacterial carbon sequestration and demonstrate the ability of chemoautotrophs to sequester carbon during sulfide mineral oxidation in mine tailings. This study is the first to investigate carbon sequestration by autotrophic groups in mine tailings through the use of isotope tracers and DNA-SIP.