Helicobacter pylori’s type IV secretion system (T4SS) is a key virulence determinant, mediating the delivery of effector proteins such as cytotoxin-associated gene A (CagA) into host epithelial cells. Despite its significance, the molecular architecture and assembly dynamics of the inner membrane core complex within the Cag-T4SS remain inadequately resolved. This study aimed to identify and characterize the direct interaction between CagF, a chaperone protein, and CagV, an inner membrane-associated component of the Cag-T4SS. Immunoprecipitation and pull-down assays confirm the physical interaction between CagF and CagV. Computational docking and molecular dynamics simulations further elucidated the binding interface, revealing a stable interaction mediated by electrostatic complementarity, hydrogen bonding, and salt bridges. Co-expression and pull-down experiments using recombinant proteins validated these findings. Notably, deletion analysis identified the critical role of the N-terminal region (residues 50-100) of CagF in CagV binding, further supporting the computational predictions. Given the essential role of CagV in CagA translocation, we hypothesize that CagF-CagV interaction may facilitate the localization of the CagF-CagA complex near the secretion channel, promoting efficient effector translocation. These findings enhance our understanding of the Cag-secretion system’s structural organization and biogenesis.