Mycobacterium colombiense is a novel member of the Mycobacterium avium complex that produces respiratory and disseminated infections in immunosuppressed patients. At present, the morphologic and genetic basis involved in the phenotypic features of M. colombiense strains are completely unknown. In this study, we demonstrate that M. colombiense strains displaying smooth morphology have an increased ability of both: biofilm formation on a hydrophobic surface and ability to slide over of motility plates. Thin-layer chromatography experiments showed that strains of M. colombiense displaying smooth colonies produce large amounts of glycolipids with a chromatographic behavior similar to glycopeptidolipids (GPLs) of M. avium. By contrast, a natural rough variant of M. colombiense (57B strain) exhibited deficiency to produce some pigmentation with age, impaired capacity to slide, to form biofilm and to produce GPLs. According to bioinformatic analyses, a gene cluster possibly involved in the GPLs biosynthesis in M. colombiense CECT 3035 is proposed; Real-time PCR and TLC experiments showed that motile conditions of culture activate the transcription of some genes predicted to be involved in the biosynthesis of M. colombiense GPLs, which correlates with the accumulation of this kind of glycolipids in the cell envelope.