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 bases 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 both to form biofilms on a hydrophobic surface and to slide over 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 that of glycopeptidolipids (GPLs) of M. avium. Conversely, we found a natural rough variant of M. colombiense (57B strain) that lacked pigmentation and showed an impaired capacity to slide, form biofilms and produce GPLs. Based on bioinformatic analyses, a gene cluster that is possibly involved in the GPL biosynthesis in M. colombiense CECT 3035 is proposed. RT-qPCR experiments showed that motile conditions of culture activate the transcription of some genes that are predicted to be involved in key enzymatic activities of the GPL biosynthesis.