<jats:title>Significance</jats:title><jats:p>Whereas most mycobacteria do not cause disease,<jats:italic>Mycobacterium tuberculosis</jats:italic>kills more than one million people each year. To better understand why<jats:italic>Mycobacterium tuberculosis</jats:italic>is virulent and to discover chemical markers of this pathogen, we compare its lipid profile with that of the attenuated but related mycobacterium,<jats:italic>Mycobacterium bovis</jats:italic>Bacillus Calmette–Guérin. This strategy identified a previously unknown<jats:italic>Mycobacterium tuberculosis</jats:italic>-specific lipid, 1-tuberculosinyladenosine, which is produced by the Rv3378c enzyme. The crystal structure of Rv3378c provides information supporting drug design to inhibit prenyl transfer. Discovery of 1-tuberculosinyladenosine provides insight into how<jats:italic>Mycobacterium tuberculosis</jats:italic>resists killing in macrophages and a new target for diagnosing tuberculosis disease.