Type IV secretion systems (T4SSs) are transporters of Gram-negative bacteria that mediate interbacterial DNA transfer, and translocation of virulence factors into eukaryotic host cells. The α-proteobacterial genus Bartonella comprises arthropod-borne pathogens that colonize endothelial cells and erythrocytes of their mammalian reservoir hosts, thereby causing long-lasting intraerythrocytic infections. The deadly human pathogen Bartonella bacilliformis holds an isolated position in the Bartonella phylogeny as a sole representative of an ancestral lineage. All other species evolved in a separate ‘modern’ lineage by radial speciation and represent highly host-adapted pathogens of limited virulence potential. Unlike B. bacilliformis, the species of the modern lineage encode at least one of the closely related T4SSs, VirB/VirD4 or Vbh. These VirB-like T4SSs represent major host adaptability factors that contributed to the remarkable evolutionary success of the modern lineage. At the molecular level, the VirB/VirD4 T4SS was shown to translocate several effector proteins into endothelial cells that subvert cellular functions critical for establishing chronic infection. A third T4SS, Trw, is present in a sub-branch of the modern lineage. Trw does not translocate any known effectors, but produces multiple variant pilus subunits critically involved in the invasion of erythrocytes. The T4SSs laterally acquired by the bartonellae have thus adopted highly diverse functions during infection, highlighting their versatility as pathogenicity factors.