M.tuberculosis (M.tb) cell wall is a major source of immunogenic antigens and secretes molecules that are Toll like receptor (TLR) agonists which are known to play an important role in the pathogenesis of mycobacterial infection. TLRs are the key receptors for the recognition of mycobacterial antigens which results in activation of macrophages. Rv3083 (mymA) is a cell wall associated protein involved in mycolic acid synthesis. In our previous study, it has been found that rRv3083 activates human macrophages (THP-1 and peripheral blood mononuclear cells) via TLR2 as demonstrated by the significant upregulation of TLR2, co-stimulatory molecules as CD40, CD80 and antigen presenting molecule HLA-DR. Here we further test whether rRv3083 modulates the gene expression profile of macrophages and induces pro-inflammatory cytokines. Macrophages were stimulated using 5µg/ml rRv3083 protein for 24hr followed by Real-Time PCR for mRNA expression levels of TLR2, TLR4 and MyD88 and supernatant were collected for ELISA. The results showed that the macrophages stimulated with rRv3083 induces pro-inflammatory cytokines and presented a five-fold increase in TLR2 and MyD88; in contrast, expression of TLR4 was unaffected in comparison with unstimulated sample. In conclusion, our data demonstrates that rRv3083 activates macrophages through the TLR2 signaling pathway.