Mycobacterium tuberculosis (Mtb) protein kinase G (PknG) is a critical protein which can inhibit the maturation of phagosome and its fusion with the lysosome, providing Mtb’s latency and survival in the host. However, its signaling pathways and functions in the human body are still unclear and needed to be further studied. In our research, we used the human protein microarray to study the proteins interacting with PknG and have found a list of proteins that are potentially related to PknG in the host, which may contribute to the infection of Mtb. Among these positive hits, we found human protein CypA has strong interaction with PknG. We further validated the interaction between PknG and CypA in vitro and in vivo and investigated its role in the infection of macrophage cells. Finally, we found that PknG can significantly down-regulate protein expression level of CypA through phosphorylation in site of T107 as well as its induction of inflammatory response through NF-κB and ERK1/2 pathways. Meanwhile, the survival of Mycobacterium smegmetis (Msm) in the macrophages with overexpressing PknG is also improved with lessen of cytokines expression. Our results provided a resourceful research base of PknG in regards to various target human proteins and offered an insightful understanding of PknG’s function in the host.