Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), remains a major human pandemic that urgently requires new diagnostic and vaccination approaches. The lipid rich envelope of Mtb consists of diverse mycolic acids that are presented by non-polymorphic CD1b molecules to donor-unrestricted germline-encoded mycolyl lipid-reactive (GEM) T cells. However, the molecular requirements governing mycolic acid antigenicity for GEM T cell receptor (TCR) are unknown. Using a large panel of synthetic mycolates, we demonstrate that structural alterations in mycolic acid meromycolate chains that are buried deeply within the CD1b protein, are antigenic determinants that influence GEM-TCR activity. We demonstrate the antigenicity of meromycolate chains both in vitro and in ex vivo T cell responses from human TB patients, and reveal marked functional differences on GEM-TCR responses. Meromycolate chain functional group type, position and stereochemistry were critical for mycolate antigenic potency. Computational simulations suggested that meromycolate chains regulate mycolate head group movement, directly modulating GEM-TCR activity. Finally, we demonstrate CD1b expression within human lung TB granulomas, indicating a role for CD1b-lipid antigen presentation at the site of infection. Our results have significant implications for the design of novel vaccines that target GEM T cells.