The widespread agricultural application of carbofuran and concomitant contamination of surface and ground waters has raised health concerns due to the reported toxic effects of this insecticide and its degradation products. Partial bacterial degradation involves carbamate hydrolysis without breakdown of the resulting phenolic metabolite. However, the capacity to mineralize the benzofuran ring has previously been reported for several sphingomonad strains and some common metabolites, including carbofuran phenol, were identified. In the current study, catabolism of the insecticide was studied with Novosphingobium sp. KN65.2, a strain isolated from a carbofuran-exposed Vietnamese soil and utilizing the compound as a sole carbon and nitrogen source. Several KN65.2 plasposon mutants with diminished or abolished capacity to degrade and mineralize carbofuran were generated and characterized. Metabolic profiling of representative mutants revealed new metabolic intermediates, in addition to the initial hydrolysis product carbofuran phenol. The promiscuous carbofuran-hydrolyzing enzyme Mcd, which is present in several bacteria lacking carbofuran ring mineralization capacity, is not encoded by the Novosphingobium sp. KN65.2 genome. An alternative hydrolase gene required for this step was not identified, but the constitutively expressed genes of the unique cfd operon, including the oxygenase genes cfdC and cfdE, could be linked to further degradation of the phenolic metabolite. A third involved oxygenase gene, cfdI, and the transporter gene cftA, encoding a TonB-dependent outer membrane receptor with potential regulatory function, are located outside the cfd cluster. This study has revealed the first dedicated carbofuran catabolic genes and provides insight in the early steps of benzofuran ring degradation.