M. tuberculosis has a cholesterol oxidizing activity that initiates sterol metabolism, which may be related to its virulence. Rv3409c of M. tuberculosis H37Rv was annotated as a putative cholesterol oxidase, an enzyme that catalyzes cholesterol modification, based on low similarities with established cholesterol oxidase genes. The Rv3409c gene was heterologously expressed in Mycobacterium smegmatis mc2 155 and purified. Using established cholesterol oxidase assays, no cholesterol oxidizing activity was detected. The M. smegmatis ortholog MSMEG1604 shares 83% amino acid identity with Rv3409c from M. tuberculosis H37Rv. The M. smegmatis MSMEG1604 transposon mutant (Myc11) oxidized and degraded cholesterol. Therefore, we concluded that Rv3409c/MSMEG1604 does not encode a cholesterol oxidase. The colony morphology of Myc11 is different from wild type as observed by light microscopy. Lipidomic analysis revealed that a single lipid, L1334, was present only in the Myc11 cell envelope. Accumulation of L1334 in the cell wall of Myc11 resulted in a rough cell morphology phenotype as compared to the smooth wild-type M. smegmatis phenotype. Sliding motility was also retarded by disruption of MSMEG1604. Complementation of the Myc11 mutant with MSMEG1604 resulted in loss of L1334 from the cell envelope. L1334 was purified from Myc11 grown to the stationary phase for identification. Exact mass measurements and thin layer chromatography experiments revealed that L1334 was similar, but not identical to known glycopeptidolipids (GPLs), which are the major surface-exposed lipids of M. smegmatis. High resolution and multidimensional mass spectrometry and 1D and 2D NMR spectroscopy indicated that L1334 is an unusual GPL that includes the glycopeptidolipid tetrapeptide core structure, two rhamnose residues, one 6-deoxytalose residue and one 3-methoxy C24 fatty acyl chain. The 6-deoxytalose moiety in L1334 was not acetylated as is typically seen in GPLs. The GPL biosynthetic genes have all been identified. Therefore, we propose that MSMEG1604/Rv3409c is indirectly involved in lipid production. Mutation of MSMEG1604 may alter metabolite, e.g. sugar concentrations, or disrupt the production of a regulatory molecule.