Rickettsial pathogens in the genera Anaplasma and Ehrlichia cause acute infection in immunologically naive hosts and are major causes of tick-borne disease in animals and humans. Immunization with purified outer membranes induces protection against acute Anaplasma marginale infection and disease, and a proteomic and genomic approach recently identified 21 proteins within the outer membrane immunogen in addition to the well-characterized major surface proteins MSP1 to MSP5. Among the newly described proteins were the type IV secretion system (TFSS) proteins VirB9, VirB10, and conjugal transfer protein (CTP). In other gram-negative bacteria, TFSS proteins form channels, facilitate secretion of molecules, and are required for intracellular survival. However, TFSS proteins have not been explored as vaccine antigens. In this study we demonstrate that in Anaplasma marginale outer membrane-vaccinated cattle, VirB9, VirB10, and CTP are recognized by serum immunoglobulin G2 (IgG2) and stimulate memory T-lymphocyte proliferation and gamma interferon secretion. VirB9 induced the greatest proliferation in CD4+ T-cell lines, and VirB9-specific CD4+ T-cell clones responded to three A. marginale strains, confirming the VirB9-specific T-cell responses are directed against epitopes in the native protein. The three TFSS proteins are highly conserved with orthologous proteins in Anaplasma phagocytophilum, Ehrlichia chaffeensis, and Ehrlichia canis. Recognition of TFSS antigens by CD4+ T cells and by IgG2 from cattle immunized with the protective outer membrane fraction provides a rationale for including these proteins in development of vaccines against A. marginale and related pathogens.