Updated project metadata. Virulence effectors secreted by Mycobacterium tuberculosis help subvert host immune mechanisms and therefore are critical for establishment of infection and pathogenesis. However, knowledge in terms of signaling mechanisms thatmodulate the secretion of virulence factors is sparse. Here using high-throughput mass-spectrometric analysis of mycobacterial secretome,phospho-proteome and phospho-secretome combined with empirical validations, we show an unprecedented regulation of mycobacterial secretion via protein phosphorylation. The intricate relationship between phosphorylation and secretion was revealed by system-level analysis of PPI network superimposed with secretome and phospho-proteome profiles. This study establishes phosphorylation as a keydeterminant of secretion of virulence factors and unveils as yet unexplored targets for therapeutic interventions.