The intracellular survival of Mycobacterium tuberculosis (Mtb) has a central role in the pathogenesis of tuberculosis. Mtb Rv2346c is a member of 6-kDa early secreted antigenic target family of proteins， which are known to inhibit the host immune responses to promote bacillary persistence in macrophages. However， the mechanism through which Rv2346c participates in Mtb pathogenesis is unclear. In the present study， recombinant Rv2346c protein was synthesized and used to treat Bacillus Calmette-Guérin (BCG)-infected macrophages. The results showed that Rv2346c inhibited the proliferation of BCG-infected macrophages and enhanced the survival of BCG in macrophages. Tumor necrosis factor-α (TNF-α) and interleukin (IL)-6 were upregulated during BCG infection but downregulated by Rv2346c. Additional experiments showed that nuclear transcription factor-κB (NF-κB) in BCG-infected macrophages induced the production of TNF-α and IL-6. In addition， miR-155 and miR-99b had a suppressive effect on NF-κB， and the expression of these miRNAs was promoted by p38. Furthermore， Rv2346c was shown to decrease the activation of NF-κB， whereas it enhanced the phosphorylation of p38 and the expression of miR-155 and miR-99b. The function of Rv2346c was also verified in Mtb-infected mice. The results showed that Rv2346c increased the observed bacterial load and lung injury and downregulated TNF-α and IL-6 in vivo. Overall， our results reveal that Rv2346c enhances mycobacterial survival in macrophages via inhibiting the production of TNF-α and IL-6 in a p38/miRNA/NF-κB pathway-dependent manner， suggesting that Rv2346c acts as a crucial virulence factor in Mtb infection and has potential use as a target for anti-tuberculosis therapy.