C. fetus subsp. fetus (Cff) and C. fetus subsp. venerealis (Cfv) are closely related but representational differential analysis of their genomes revealed a Type IV Secretion System (T4SS) unique for Cfv. Macromolecular transporters of this type are involved in conjugative DNA transfer and/or delivery of effector proteins to host cells, thereby contributing to bacterial virulence. The T4SS is part of a 42 kb pathogenicity island (PAI) located on the chromosome which also encodes two putative effector proteins having an FIC-domain. Conjugation experiments revealed that plasmid DNA was mobilized from Cfv to other bacteria in a process requiring both T4SS genes virB9 and virD4. Complementation of DNA delivery in trans was temperature and promoter dependent.We hypothesized that interbacterial DNA transfer is unlikely to be the only function for the T4SS and propose that the system also translocates proteins, specifically the putative FIC effector proteins, to eukaryotic host cells. Since little is known about C. fetus virulence mechanisms, we adapted in vitro approaches typical for other Campylobacter to evaluate host-pathogen interactions. Gentamicin protection assays and immunofluorescence staining revealed that both C. fetus subspecies invade a variety of eukaryotic cells, including human intestinal cells (CaCo-2) and human placenta cells (ACH-3P). Additionally, we demonstrated that C. fetus migrates underneath the cultured cells, in good agreement with a proposed virulence strategy of Campylobacter. Clinical Cfv isolates generally exhibited higher invasion levels in cell culture compared to Cfv ATCC 19438 (CfvT) and comparable to C jejuni. Inactivation of virD4 in the sequenced Cfv 84-112 significantly reduced (50%) invasion of CaCo-2 cells. Complementation of this phenotype by vir gene expression in trans links virulence associated properties to the T4SS.