Tuberculosis (TB), caused by Mycobacterium tuberculosis, remains a formidable threat to global public health. The successful intracellular persistence of M. tuberculosis significantly contributes to the intractability of tuberculosis. Proline-glutamic acid (PE) and proline-proline-glutamic acid (PPE) are mycobacterial exclusive protein families that widely reported to be involved in the bacterial virulence, physiology and interaction with host. Rv2770c (PPE44), a predicted virulence factor, was up-regulated upon the infected guinea pig lungs. To investigate the role of Rv2770c, we heterologously expressed the PPE44 in the nonpathogenic fast-growing M. smegmatis strain. Subcellular location analysis demonstrated that Rv2770c is a cell wall associated protein, suggestive of a potential candidate involved in host-pathogen interaction. The Rv2770c can enhance M. smegmatis survival within macrophages and under stresses such as H2O2, SDS, diamide exposure, and low pH condition. M. smegmatis expressing Rv2770c is more virulent as testified by the increased death of macrophages and the increased expression of interlukin-6 (IL-6) and interlukin-12p40 (IL-12p40). Moreover, Rv2770c altered the secretion of IL-6 and IL-12p40 of macrophages via NF-κB, ERK1/2 and p38 MAPK axis. Taken together, this study implicated that Rv2770c was a virulent factor actively engaged in the interaction with host macrophage.