Given the enormous health burden of Tuberculosis (TB), great effort has been invested into understanding the molecular mechanisms governing mycobacterial pathogenesis. The ability of Mycobacterium tuberculosis (Mtb), the primary causative agent of TB, to colonise and survive within a host is dependent on a highly complex host-pathogen interplay. The bacterial mechanisms that underpin this interplay are guided by a myriad of genes whose function, ranging from structural and enzymatic proteins to protein secretion systems, are necessary to resist host defence mechanisms, permit access to new tissue sites, and facilitate transmission to a naive host. The timely expression of these genes within correct environment is critical for successful infection, and as such Mtb requires sensitive, responsive systems of gene regulation to efficiently perform this function. Two-component systems (TCS) are signal transduction pathways comprised of a sensor kinase and a response regulator that function to allow pathogenic organisms, including Mtb, to respond appropriately to environments encountered within a host. We sought to characterise a novel mycobacterial TCS, LirAB, and understand its impact on genetic regulation during infection. Transcriptional analysis demonstrates that LirAB responds to low pH, a key Mtb intra-macrophage stressor, and is upregulated during infection of THP-1 human monocytes and murine bone-marrow derived macrophages. We demonstrate that this 2CR regulates the expression of several pH-associated virulence and persistence determinants of Mtb infection, and show that genetic disruption of this 2CR attenuates its fitness in an in vivo murine model of TB. Our work suggests that the LirAB 2CR is an important virulence regulator of Mtb and highlights the importance of environmental sensing on the regulation of Mtb infection pathways.