Cag type IV secretion system (CagT4SS) mediates the translocation of a wide spectrum of substrates including oncoprotein CagA to host cells and plays an indispensable role in the pathogenesis of Helicobacter pylori. CagM, one of the central cylinder components responsible for initiating the assembly of CagT4SS, is able to undergo high-order homo-oligomerization. Two CagM mutants, NTD-EK (E29K/K31E/K32E/E33K/E35K/E36K) and DEK (D183E/E184K/E185K), deficient in high-order oligomerization were generated. Here, we performed differential HDX-MS analysis on wild-type, to characterize the interface involved in homo-oligomerization. Our findings reveal the significance of CagM homo-oligomerization on facilitating CagT4SS assembly.