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Skatole is a typical malodor compound in animal wastes. Several skatole-degrading bacterial strains have been obtained, whereas the molecular response of strains to skatole stress has not been well elucidated. Herein, the skatole degradation by a Gram-positive strain Rhodococcus aetherivorans DMU1 was investigated. Strain DMU1 showed high efficiency in skatole degradation under the conditions of 25–40 °C and pH 7.0–10.0. It could utilize various aromatics, including cresols, phenol, and methylindoles, as the sole carbon source for growth, implying its potential in the bioremediation application of animal wastes. Transcriptomic sequencing revealed that 328 genes were up-regulated and 640 genes were down-regulated in strain DMU1 when grown in the skatole-containing medium. Skatole increased the gene expression levels of antioxidant defense systems and heat shock proteins. The expression of ribosome-related genes was significantly inhibited which implied the growth inhibition of skatole. A rich set of oxidoreductases were changed, and a novel gene cluster containing the flavoprotein monooxygenase and ring-hydroxylating oxygenase genes was highly up-regulated, which was probably involved in skatole upstream degradation. The upregulation pattern of this gene cluster was further verified by qRT-PCR assay. Furthermore, skatole should be mainly degraded via the catechol ortho-cleavage pathway with cat25170 as the functional gene. The gene cat25170 was cloned and expressed in E. coli BL21(DE3). Pure enzyme assays showed that Cat25170 could catalyze catechol with Km 9.96 μmol/L and kcat 12.36 s−1.