Type IV secretion systems (T4SSs) are transport machineries of Gram-negative bacteria that mediate interbacterial DNA-transfer, and secretion of virulence factors into eukaryotic target cells. A growing number of human pathogenic bacteria use T4SSs for intercellular delivery of effector molecules that modify host cellular functions in favour of the pathogen. Recent advances in studying the molecular mechanisms of Bartonella pathogenesis have provided evidence for the central roles of two distinct T4SSs, VirB/VirD4 and Trw, in the ability of the bacteria to colonize, invade and persist within either vascular endothelial cells or erythrocytes, respectively. The identification of VirB/VirD4-transported substrates and the delineation of their secretion signal have paved the way towards understanding the molecular mechanisms underlying Bartonella-host cell interaction and modulation, as well as the exploitation of this system for engineered substrate delivery into mammalian target cells.