Root exudates shape microbial communities at the plant-soil interface. Here we compared bacterial communities that utilize plant-derived carbon in the rhizosphere of wheat in different soil depths, including topsoil, as well as two subsoil layers up to 1 m depth. The experiment was performed in a greenhouse using soil monoliths with intact soil structure taken from an agricultural field. To identify bacteria utilizing plant-derived carbon, C-CO labelling of plants was performed for two weeks at the EC50 stage, followed by isopycnic density gradient centrifugation of extracted DNA from the rhizosphere combined with 16S rRNA gene-based amplicon sequencing. Our findings suggest substantially different bacterial key players and interaction mechanisms between plants and bacteria utilizing plant-derived carbon in the rhizosphere of subsoils and topsoil. Among the three soil depths, clear differences were found in C enrichment pattern across abundant operational taxonomic units (OTUs). Whereas, OTUs linked to Proteobacteria were enriched in C mainly in the topsoil, in both subsoil layers OTUs related to Cohnella, Paenibacillus, Flavobacterium showed a clear C signal, indicating an important, so far overseen role of Firmicutes and Bacteriodetes in the subsoil rhizosphere.