Aims: To determine whether resuscitation-promoting factor (RPF) from Mycobacterium tuberculosis can promote mycobacterial growth and shorten culture time. Method and Results: We cloned, expressed and purified an RPF from M. tuberculosis, Rv1009 protein and subsequently studied the biological activity of the recombinant Rv1009 (rRv1009) in liquid and on solid media. Our results indicate that the molecular weight of rRv1009 protein expressed in Escherichia coli BL21 was approximately 39 kDa. At picomolar and micromolar concentrations, rRv1009 protein could increase the optical density of freeze-dried Mycobacterium bovis BCG three to fivefold in Middlebrook 7H9 medium, stimulate the growth of viable mycobacteria on solid medium, and shorten positive growth detection time of a small number of M. tuberculosis in BACTEC 960 medium. Conclusions: The rRv1009 could promote proliferation of mycobacteria. It may be useful for culture of mycobacteria presented in clinical samples. Significance and Impact of the Study: rRv1009 protein can be used as a growth-promoting reagent of mycobacteria in the medium to shorten the time of culture.