Biobeds, designed to minimize pesticide point source contamination, mainly rely on biodegradation processes. We studied the interactions of a biobed microbial community with the herbicide isoproturon (IPU) to explore the role of the pdmA gene, encoding the large subunit of an N-demethylase responsible for the initial demethylation of IPU via qPCR and RT-qPCR and the effect of IPU on the diversity of the total bacterial community and its active fraction through amplicon sequencing of DNA and RNA, respectively. We further investigated the localization and dispersal mechanism of pdmAB in the biobed packing material by measuring the abundance of the plasmid pSH (harboring pdmAB) of the IPU-degrading Sphingomonas sp. SH strain (previously isolated from the soil used in the biobed) compared with the abundance of the pdmA gene and metagenomic fosmid library screening. pdmA abundance and expression increased concomitantly with IPU mineralization verifying its major role in IPU transformation in the biobed system. DNA- and RNA-based 16S rRNA gene sequencing analysis showed no effects on bacterial diversity. The pdmAB-harboring plasmid pSH showed consistently lower abundance than pdmA, suggesting the localization of pdmAB in replicons other than pSH. Metagenomic analysis identified four pdmAB-carrying fosmids. In three of those, pdmAB were organized in a well-conserved operon carried by sphingomonads' plasmids with low synteny to pSH, while the fourth fosmid contained an incomplete pdmAB cassette localized in a genomic fragment of a Rhodanobacter strain. Further analysis suggested a potential crucial role of IS6 and IS256 in the transposition and activation of the pdmAB operon.Importance: Our study provides novel insights in the interactions of IPU with the bacterial community of biobed systems, reinforces the assumption of a transposable nature of IPU-degrading genes and verifies that on-farm biobed systems are hotspots for the evolution of pesticide catabolic traits.