CXCL10 production is a critical step in limiting mycobacterial infection. Although induction of this chemokine by mycobacteria in epithelial cells has been reported, it is still unclear how CXCL10 is regulated in Mycobacterium bovis BCG-infected epithelial cells. In this study, we demonstrate that phosphatidylinoditol 3-kinase (PI3K)/Akt and the nuclear factor kB (NF-kB) signaling pathways play an important role in CXCL10 expression at the protein and mRNA level in A549 cells. We demonstrate that treatment of A549 cells with LY294002 and wortmannin, two PI3K inhibitors, inhibited M. bovis BCG-induced CXCL10 expression. In addition, treatment of A549 cells with an Akt inhibitor significantly blocked M. bovis BCG-induced CXCL10 production. Moreover, our data show that treatment of epithelial cells with CAPE, BAY 11-7082, and PDTC three selective inhibitors of NF-kB, significantly reduced the effect of M. bovis BCG on induced CXCL10 mRNA expression (74%, 69% and 83% inhibition by 8microM CAPE, 10microM BAY 11-7082 and 3microM PDTC as assessed by real-time PCR, respectively). In accordance with the gene induction, the production of CXCL10 was also significantly reduced by these inhibitors. Finally, the inhibition of PI3K affect NF-kB activation in M. bovis BCG-infected cells, indicating that PI3K activity is required for the M. bovis BCG-induced activation of NF-kB. The functional association between PI3K/Akt and NF-kB demonstrates another mechanism in the regulation of M. bovis BCG-induced CXCL10 in A549 cells.