Polychlorinated dibenzo-p-dioxins (PCDDs) are released into the environment from a variety of both anthropogenic and natural sources. While highly chlorinated dibenzo-p-dioxins are persistent under oxic conditions, in anoxic environments, these organohalogens can be reductively dechlorinated to less chlorinated compounds that are then more amenable to subsequent aerobic degradation. Identifying the microorganisms responsible for dechlorination is an important step in developing bioremediation approaches. In this study, we demonstrated the use of a DNA-stable isotope probing (SIP) approach to identify the bacteria active in dechlorination of PCDDs in river sediments, with 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-TeCDD) as a model. In addition, pyrosequencing of reverse transcribed 16S rRNA of TeCDD dechlorinating enrichment cultures was used to reveal active members of the bacterial community. A set of operational taxonomic units (OTUs) responded positively to the addition of 1,2,3,4-TeCDD in SIP microcosms assimilating 13C-acetate as the carbon source. Analysis of bacterial community profiles of the 13C labeled heavy DNA fraction revealed that an OTU corresponding to Dehalococcoides mccartyi accounted for a significantly greater abundance in cultures amended with 1,2,3,4-TeCDD than in cultures without 1,2,3,4-TeCDD. This implies the involvement of this Dehalococcoides mccartyi strain in the reductive dechlorination of 1,2,3,4-TeCDD and suggests the applicability of SIP for a robust assessment of the bioremediation potential of organohalogen contaminated sites.