Membrane peptides appear as an emerging class of regulatory molecules in bacteria, which can interact with membrane proteins, such as sensor kinases. To date, regulatory membrane peptides have been completely overlooked in mycobacteria. The 30 amino-acid-long KdpF peptide, which is co-transcribed with kdpABC genes and regulated by the KdpDE two-component system, is supposed to stabilize the KdpABC potassium transporter complex but may also exhibit unsuspected regulatory function(s) towards the KdpD sensor kinase. Herein, we showed by quantitative RT-PCR that the Mycobacterium bovis BCG kdpAB and kdpDE genes clusters are differentially induced in potassium-deprived broth medium or within infected macrophages. We have overexpressed the kdpF gene in M. bovis BCG to investigate its possible regulatory role and effect on mycobacterial virulence. Our results indicate that KdpF does not play a critical regulatory role on kdp genes expression despite the fact that KdpF interacts with the KdpD sensor kinase in a bacterial two-hybrid assay. However, overexpression of kdpF results in a significant reduction of M. bovis BCG growth in both murine and human primary macrophages, and is associated with a strong alteration of colonial morphology and impaired cording formation. To identify novel KdpF interactants, a mycobacterial library was screened using KdpF as bait in the bacterial two-hybrid system. This allowed us to identify members of the MmpL family of membrane proteins, known to participate in the biosynthesis/transport of various cell wall lipids, thus highlighting a possible link between KdpF and cell wall lipid metabolism. Taken together, these data suggest that KdpF overexpression reduces intramacrophage growth which may result from alteration of the mycobacterial cell wall.