Indole, an important signaling molecule as well as a typical N-heterocyclic aromatic pollutant, is widespread in nature. However, the biotransformation mechanisms of indole are still poorly studied. Here, we sought to unlock the genetic determinants of indole biotransformation in strain Cupriavidus sp. SHE based on genomics, proteomics and functional studies. A total of 177 proteins were notably altered (118 up- and 59 downregulated) in cells grown in indole mineral salt medium when compared with that in sodium citrate medium. RT-qPCR and gene knockout assays demonstrated that an indole oxygenase gene cluster was responsible for the indole upstream metabolism. A functional indole oxygenase, termed IndA, was identified in the cluster, and its catalytic efficiency was higher than those of previously reported indole oxidation enzymes. Furthermore, the indole downstream metabolism was found to proceed via the atypical CoA-thioester pathway rather than conventional gentisate and salicylate pathways. This unusual pathway was catalyzed by a conserved 2-aminobenzoyl-CoA gene cluster, among which the 2-aminobenzoyl-CoA ligase initiated anthranilate transformation. This study unveils the genetic determinants of indole biotransformation and will provide new insights into our understanding of indole biodegradation in natural environments and its functional studies.