As a typical brominated flame retardants (BFRs), 2,4,6-tribromophenol (TBP) has serious hazard to the environmental health and its environmental fate has attracted considerable attention. Dehalogenation reaction plays key role in microbial TBP degradation and detoxification. So far, several halophenols-degrading enzymes have been reported to transform their substrate by oxidative dehalogenation; however, the molecular and biochemistry characterization of microbial hydrolytic dehalogenation is limited. In this study, Cupriavidus sp. CNP-8 with high TBP degradation activity was found to degrade TBP via an obviously differnet pathway as compared to other reported TBP-degraders. The transcription of hnp genes were significantly upregulated with TBP stimulation, indicating their involvment in TBP degradation. Enzymatic assays with ¹⁸O-labeling experiments showed that HnpAB, a two-component FAD-dependent monooxygenase, transformed TBP via consecutive oxidative and hydrolytic debromination reactions with the formation of 6-bromo-1,2,4-benzenetriol (BBT) as the ring-cleavage substrate. The function of the BBT ring-cleavage enzyme (HnpC) was also characterized both in vitro and in vivo. This finding provides new molecular mechanism of microbial detoxification of TBP and novel information of the environmental fate of this BFRs. Furthermore, to investigate the frequency of this novel dehalogenation mechanism in microbes, we also analyzed the distribution as well as the genetic structure of the hnpABC cluster by comparative genomics. Although hnpA homolog is distributed in several bacterial genera including Cupriavidus, Paraburkholderia, Variovorax and Streptomyces, the complete hnpABC cluster is only retrieved from Cupriavidus and strictly conservative in the genomes. This indicated that Cupriavidus have unique evolutionary pattern in acquiring the hnpABC to degrade TBP and its analogs, enhancing our understanding of the microbial adaptive evolution in halophenols-contaminated environment.