Denitrification-based remediation has been proved as a cost-effective approach for organic removal in sediment. However, little attention has been drawn on the concomitant autotrophic denitrification process and its impacts during such treatment. In this study, a contaminated marine sediment sample was treated with nitrate in a series of experiments to characterize the autotrophic denitrification and its impacts on metal speciation. Through treatment, as the consequence of autotrophic denitrification which accounts for 73.9% of nitrate reduction, approximately 98.8% acid volatile sulfide (AVS) was oxidized to sulfate, causing changes of Zn, Cu and Pb speciation in the sediment. Their oxidizable fractions decreased by 71.7%, 13% and 71% respectively while the bound-to-carbonate fractions increased by 52.0%, >700% and >40%, and the reducible fractions also increased by 276%, >280% and 140%. Thus, the relatively stable oxidizable phase of Zn, Cu and Pb was generally transferred to the more mobile bound-to-carbonate and reducible phases. According to SEM (simultaneously extracted metal) analysis, most of extractable Zn and Pb were no longer present in the form of metal sulfides after denitrification. The (Zn+Pb)/AVS ratio increased from 0.030 to 3.1. Both sequential extraction and AVS-SEM suggested a possible increase of heavy metal mobility and, thus, toxicity. Two major species responsible for autotrophic denitrification were identified to be phylogenetically related with Sulfurimonas paralvinellae and Thiohalophilus thiocyanoxidans.