In this study, we utilized a multi-omic approach, encompassing proteomics, phosphoproteomics, and interactomics, to examine the influence of mycobacterial PknG-mediated regulation on host cellular processes. Our findings suggest that host lysyl-tRNA synthetase (KARS) is a potential substrate of PknG within host cells and may modulate immune response injury by affecting the phosphorylation levels of the MAPKs/NF-κB signaling pathway. Furthermore, our research demonstrates that mycobacterial PknG specifically catalyzes the phosphorylation of KARS at the T592/T595 sites, which can suppress TNF-α secretion from immune cells. Our study revealed that the PknG-KARS phosphorylation signaling axis influences the reduction of inflammatory factors in macrophages.