Objective To compare the stress tolerance of recombinant Mycobacterium smegmatis strains Ms-PPE61 and Ms-Vec under different external stress conditions, investigate the activation/inhibition levels of the mitogen-activated protein kinase (MAPK)/nuclear factor (NF)-κB signaling pathway following their infection of macrophages, and explore differences in inflammatory cytokine expression after infection of RAW264.7 cells. Methods Ms-Vec and Ms-PPE61 were constructed and cultured to the logarithmic growth phase before being subjected to acidic, SDS, and H₂O₂ conditions. Colony-forming units (CFUs) were measured at different time points. Proteins were extracted from cells collected 1-48 h post-infection (hpi), and the expression levels of signaling pathway marker molecules were determined by Western blotting. The interleukin (IL)-6, tumor necrosis factor (TNF)-α, and IL-1β concentrations in the supernatants of RAW264.7 cells infected with Ms-Vec and Ms-PPE61 were measured by ELISA at 24 hpi and 48 hpi. GraphPad Prism 7.0 was used for analysis of variance of the data, and P<0.05 was considered significant. Results PCR revealed the presence of a target band in Ms-PPE61 but not in Ms-Vec. Coomassie brilliant blue staining confirmed consistent protein loading. Western blotting showed that Ms-PPE61 expressed a ~42 kDa Flag fusion protein, while Ms-Vec did not. Ultra-high-speed centrifugation was performed to separate the components of M. smegmatis. Western blotting revealed that the cytoplasmic marker protein GroES was expressed in the cytoplasmic fractions of both Ms-Vec and Ms-PPE61, while the Flag-tagged target protein was exclusively present in the cell wall of Ms-PPE61. After treatment under acidic conditions (pH 3.0) for 3 h, the survival rate of Ms-PPE61 was higher than that of Ms-Vec (P<0.000 1), while the survival rate showed no significant difference after treatment for 6 h and 9 h (P>0.05). After treatment with 0.2% SDS for 3, 6, and 9 h, the survival rate of Ms-PPE61 was higher than that of Ms-Vec (P<0.000 1). Similarly, after H₂O₂ treatment for 3 h and 6 h, the survival rate of Ms-PPE61 was higher than that of Ms-Vec (P<0.000 1). Western blotting showed that the Ms-PPE61 group had significantly lower p-p38 and p-ERK levels at 48 hpi and higher IκB-α levels at all time points than the Ms-Vec group. ELISA results indicated no differences in TNF-α secretion between the Ms-PPE61 and Ms-Vec groups at 24 hpi and 48 hpi (P>0.05), while the Ms-PPE61 group had lower IL-6 levels at 24 hpi and 48 hpi (P<0.000 1) and lower IL-1β level at 48 hpi (P<0.01) than the Ms-Vec group. Conclusion PPE61 can enhance the tolerance of recombinant Mycobacterium smegmatis to acidic, SDS and H₂O₂ stress, inhibit the MAPK and NF-κB signaling pathways by down-regulating the expression of p-p38 and p-ERK and up-regulating the expression of IκB-α, and reduce the secretion of IL-6 (significantly at both 24 h and 48 h) and IL-1β (significantly at 48 h) in macrophages, but has no significant effect on the secretion of TNF-α.