The human pathogen Helicobacter pylori is involved in gastric diseases ranging from gastritis to gastric cancer. Virulent strains harboring the cag pathogenicity island (cag PAI) which encode a Type IV Secretion System (T4SS) can induce pro-inflammatory cytokines such as interleukin-8 and deliver their major effector proteins CagA into the gastric cells. While a subset of cag PAI genes have been identified to be the homologues of T4SS genes from Agrobacterium tumefaciens, a majority have unknown functions. We have identified one of such proteins, Cag1, which was predicted to be a non-classically secreted and virulent protein. Our results showed that Cag1 is a membrane-associated protein essential for the induction of multiple cytokine secretions, and cag1-deficient mutant has partial influence on CagA translocation; while the protein itself was not injected into host cells. Our data indicated that Cag1 is located in the bacterial membrane and is associated with the function of T4SS.