Ribosomal-protein-alanine acetyltransferase (RimI) is a member of GCN5-related N-acetyltransferase family of Mycobacterium tuberculosis (M.tb) which is encoded by rimI gene. The biological function of RimI in M．tb is still not clear. In order to explore the biological characteristics of RimI and its role in the virulence of M．tb, Mycobacterium smegmatis (M.sm) was selected as a model strain to construct a recombinant strain Msm∷pMV261-rimI. The difference in growth rate, colony morphology, and biofilm formation between Msm∷pMV261 and Msm∷pMV261-rimI were analyzed in vitro. Their resistance to hypoxia, acidic pH, dithiothreitol (DTT), H₂O_2, sodium dodecyl sulfate (SDS) and intracellular survival ability during the infection of macrophages in Raw264.7 cell line were explored. Compared with the Msm∷pMV261 strain, the growth curve and biofilm forming of the Msm∷pMV261-rimI strain were slowed down in the early and middle growth stages, but the late maturity of biofilm was not affected. Moreover, the Msm∷pMV261-rimI strain not only increased its resistance to acidic or hypoxic stress, its intracellular survival ability was also enhanced during the infection of macrophages in Raw264.7 cell line. Altogether, the results indicated that the rimI gene played an important role in biofilm formation, stress resistance and intracellular survival of M．tb, suggesting that rimI gene may be a virulence factor related to the pathogenicity of M．tb.