The success of Mycobacterium tuberculosis (M. tuberculosis) as a pathogen is largely contributes to its ability to manipulate the host immune responses. The genome of M. tuberculosis encodes multiple immune-modulatory proteins, including several members of the multi-genic PE_PPE family. Despite of intense research, the roles of PE_PGRS proteins in mycobacterial pathogenesis remain elusive. The function of M. tuberculosis PE_PGRS41, characterized by an extended and unique C-terminal domain, was studied. Expression of PE_PGRS41 in Mycobacterium smegmatis, a non-pathogenic species intrinsically deficient of PE_PGRS, severely impaired the resistance of the recombinant to multiple stresses via altering the cell wall integrity. Macrophages infected by M. smegmatis harboring PE_PGRS41 decreased the production of TNF-α, IL-1β and IL-6. In addition, PE_PGRS41 boosted the survival of M. smegmatis within macrophage accompanied with enhanced cytotoxic cell death through inhibiting the cell apoptosis and autophagy. Taken together, these results implicate that PE_PGRS41 is a virulence factor of M. tuberculosis and sufficient to confer pathogenic properties to M. smegmatis.