Background: Precise chemical structure and biosynthetic pathway of the acyl chain of mycobacterial glycopeptidolipids (GPLs) is unknown. Results: Polyketide synthases dictate biosynthesis and determine hydroxylation at C-5 position of the GPL acyl chain. Conclusions: Retrobiosynthetic studies establish the role of bimodular polyketide synthase and fatty acyl-AMP-ligase in GPL biosynthesis. Significance: The long-standing ambiguity on the accurate structure and the biosynthetic mechanism of GPLs was resolved.