The purpose of the current study is to investigate the physiological effects in Mycobacterium smegmatis (M. smegmati) caused by the overexpression of GroEL1, which is a heat shock protein in Mycobacterium tuberculosis. Bacterial morphous was observed using transmission electron microscope (TEM), and bacterial population growth curve was plotted. Drug susceptibility of recombinant M. smegmatis was assayed using serial dilution. BALB/c mice infected by recombinant M. smegmatis were used as model to detect bacterial viability in vivo and virulence changes related to GroEL1 overexpression. As GroEL1 overexpressed, the level of bacteria bacterial ribosome increased significantly, so did the population growth and adaptive capacity. But for the resisrance to commonly used first-line and second-line anti-tuberculosis drugs did not change. The tesults showed that overexpression of GroEL could help M. smegmatis extend life cycle in hosts, and enhance bacterial virulence, with a significant impact on growth of bacteria in vivo or in vitro.