Infection with Mycobacterium tuberculosis (Mtb), the bacterium that causes tuberculosis, remains a global health concern. Both classically and non-classically restricted cytotoxic CD8+ T cells are important to the control of Mtb infection. We and others have demonstrated that the non-classical MHC I molecule HLA-E can present pathogen-derived peptides to CD8+ T cells. In this manuscript, we identified the antigen recognized by an HLA-E-restricted CD8+ T cell clone isolated from an Mtb latently infected individual as a peptide from the Mtb protein, MPT32. Recognition by the CD8+ T cell clone required N-terminal O-linked mannosylation of MPT32 by a mannosyltransferase encoded by the Rv1002c gene. This is the first description of a post-translationally modified Mtb-derived protein antigen presented in the context of an HLA-E specific CD8+ T cell immune response. The identification of an immune response that targets a unique mycobacterial modification is novel and may have practical impact in the development of vaccines and diagnostics.