Deoxynivalenol (DON) is frequently detected in cereals and cereal-based products and has a negative impact on human and animal health. In this study, an unprecedented DON-degrading bacterial isolate D3_3 was isolated from a sample ofTenebrio molitorlarva feces. A 16S rRNA-based phylogenetic analysis and genome-based average nucleotide identity comparison clearly revealed that strain D3_3 belonged to the speciesKetogulonicigenium vulgare. This isolate D3_3 could efficiently degrade 50 mg/L of DON under a broad range of conditions, such as pHs of 7.0–9.0 and temperatures of 18–30 °C, as well as during aerobic or anaerobic cultivation. 3-keto-DON was identified as the sole and finished DON metabolite using mass spectrometry. In vitro toxicity tests revealed that 3-keto-DON had lower cytotoxicity to human gastric epithelial cells and higher phytotoxicity toLemna minorthan its parent mycotoxin DON. Additionally, four genes encoding pyrroloquinoline quinone (PQQ)-dependent alcohol dehydrogenases in the genome of isolate D3_3 were identified as being responsible for the DON oxidation reaction. Overall, as a highly potent DON-degrading microbe, a member of the genusKetogulonicigeniumis reported for the first time in this study. The discovery of this DON-degrading isolate D3_3 and its four dehydrogenases will allow microbial strains and enzyme resources to become available for the future development of DON-detoxifying agents for food and animal feed.