Updated project metadata. Tuberculosis (TB), caused by Mycobacterium tuberculosis (Mtb), is a leading cause of infectious disease mortality worldwide for which no sufficiently protective vaccine exists. CD8+ T cells contribute to protective immunity to TB, but the antigenic targets presented on MHC-I by macrophages infected with virulent Mtb for recognition by CD8+ T cells have not been conclusively defined. Here, we use mass spectrometry to directly identify Mtb-derived peptides presented on MHC class I (MHC-I) by infected primary human monocyte-derived macrophages. We identified 16 MHC-I peptides derived from 12 Mtb proteins, and validated these identifications using internal standard parallel reaction monitoring (SureQuant). Our results show that this set of antigens is highly enriched for substrates of type VII secretion systems (T7SS) relative to the Mtb proteome. Our results identify specific Mtb proteins that may be suitable targets for subunit vaccines designed to elicit protective CD8+ T cell-mediated immunity and suggest that T7SS substrates may be a fruitful class of antigens to prioritize for further vaccine target discovery efforts. Note: donors labeled A, C, D, E, H, and I in file names here are renamed sequentially (A, B, C, D, E, and F) in the manuscript describing this study to avoid confusion.