The proline-proline-glutamic acid (PPE) family proteins are abundant only in pathogenic Mycobacteria, but their general functions are far from unveiled. To investigate their roles in how Mycobacterium tuberculosis (Mtb) resists killing by the host, 25 PPE recombinant Mycobacterium smegmatis strains that overexpress Mtb PPE proteins were constructed. During phagocytosis, a similar amount of intracellular bacteria was observed at 2 h post-infection (hpi) for 24 PPE recombinants, while a 50% reduction of entrance was observed for the PPE29 recombinant. In addition, we found that 20 ppe genes significantly influenced the survival of mycobacteria within macrophage cells. Mycobacterial survival was promoted by overexpression of 18 of these genes and inhibited by the other two. Highest survival was observed for the PPE27 recombinant. We also measured the levels of proinflammatory cytokines tumor necrosis factor-alpha and interleukin-6 secreted by macrophages. The overall effects varied among the different PPE recombinants. Moreover, we also found that various PPE recombinants exhibited increased resistance against oxidative, acidic and sodium dodecyl sulfate stresses that could be encountered in vivo. Together, our results indicate that PPE proteins play distinct roles in mycobacterial survival in macrophages. The findings described here broaden our understanding of mycobacterial pathogenicity.