The taxonomic position of the cholesterol-degrading strain Chol-3(T), isolated from a sewage sludge sample, was clarified using a polyphasic taxonomic approach. Phylogenetic analysis of its 16S rRNA gene sequence, whole-cell fatty acid profile and mycolic acid composition revealed that this isolate is a member of the genus Gordonia with the species Gordonia sihwensis, G. hydrophobica and G. shandongensis being the nearest phylogenetic neighbours. The results of DNA-DNA hybridization against its phylogenetically closest neighbours as well as the results of physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain Chol-3(T) from the other Gordonia species with validly published names. Strain Chol-3(T) therefore merits recognition as a member of a novel species within the genus Gordonia, for which the name Gordonia cholesterolivorans sp. nov. is proposed. The type strain is Chol-3(T) (=CECT 7408(T) =DSM 45229(T)).