Rv3425 encodes the PE/PPE family protein PPE57, which is a candidate antigen for vaccine construction that was tested in mice and rhesus monkeys in early studies of this laboratory. To study the function of Rv3425 of Mycobacterium tuberculosis (M. tuberculosis), Mycobacterium smegmatis (M. smegmatis) was selected as a model strain, and the recombinant strain Ms-Rv3425 was constructed. Compared with wild type, the colony morphology of Ms-Rv3425 was rougher and more bulging, and the ability of pellicle and aggregate formation was stronger. Ms-Rv3425 showed higher resistance to stress when cultured in acidic, sodium dodecyl sulfate (SDS), ampicillin, isoniazid, and rifampin conditions. The expression level of Rv3425 in the attenuated strain of M. tuberculosis H37Ra cultured under these adverse conditions was also significantly up-regulated. Infection of macrophages showed that Ms-Rv3425 enhanced the intracellular survival of M. smegmatis in THP-1 cell line. It led to higher mortality in BALB/c mice, and the bacterial retention and pathological damage of each organ were also higher than that of the control group. In summary, over expression of Rv3425 in M. smegmatis enhanced stress resistance, drug resistance and virulence.