Abstract: Objective To determine how Csm4 protein expression affects intracellular survival of Mycobacterium smegmatis(MS). Methods Csm 4 gene was amplified by PCR to construct pMV261-Csm4 shuttle expression plasmid. The Csm4 protein expression in MS_Csm4 was detected by Western blot after electroporation of the recombinant plasmid into MS. The growth kinetics of MS_Csm4 in vitro and the influence of reactive N, O species on the growth of MS_Csm4were observed. The intracellular survival of MS_Csm4 and expressions of inducible nitric oxide synthase gene (iNOS) and nitric oxide production (NO) were detected after infection with THP-1 macrophages. Results Csm4 protein was successfully expressed in MS_Csm4, which did not affect the growth of the recombinant MS. Reactive N, O species decreased MS_Csm4 colony forming unit (CFU) in vitro. THP-1 increased the expression of iNOS and NO production and decreased intracellular survival of MS_Csm4. Conclusion Recombinant MS_Csm4 is susceptible to reactive N, O species in vitro. THP-1 promotes NO release and thus discourages intracellular survival of MS.