A specificity of Brassicaceous plants is the production of sulphur secondary metabolites called glucosinolates that can be hydrolysed into glucose and biocidal products. Among them, isothiocyanates are toxic to a wide range of microorganisms and particularly soil-borne pathogens. The aim of this study was to investigate the role of glucosinolates and their breakdown products as a factor of selection on rhizosphere microbial community associated with living Brassicaceae. We used a DNA-stable isotope probing approach to focus on the active microbial populations involved in root exudates degradation in rhizosphere. A transgenic Arabidopsis thaliana line producing an exogenous glucosinolate and the associated wild-type plant associated were grown under an enriched (13)CO(2) atmosphere in natural soil. DNA from the rhizospheric soil was separated by density gradient centrifugation. Bacterial (Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria and Acidobacteria), Archaea and fungal community structures were analysed by DGGE fingerprints of amplified 16S and 18S rRNA gene sequences. Specific populations were characterized by sequencing DGGE fragments. Roots of the transgenic plant line presented an altered profile of glucosinolates and other minor additional modifications. These modifications significantly influenced microbial community on roots and active populations in the rhizosphere. Alphaproteobacteria, particularly Rhizobiaceae, and fungal communities were mainly impacted by these Brassicaceous metabolites, in both structure and composition. Our results showed that even a minor modification in plant root could have important repercussions for soil microbial communities.