Coxiella burnetii, the causative agent of human Q fever, is an intracellular pathogen that replicates in a lysosome-derived vacuole. The molecular mechanisms used by this bacterium to create this unique replicative niche remain largely unknown. Advances in axenic culture and genetic manipulation of this organism have allowed us to conduct a large scale visual screen of a C. burnetii transposon-insertion mutant library to identify genes required for biogenesis of a mature Coxiella-containing vacuole (CCV). This study confirmed our earlier findings that the Dot/Icm Type IV Secretion System is essential for intracellular replication of C. burnetii and also allowed identification of individual Dot/Icm effectors that are required for development of the CCV. Disruption of the gene encoding the effector Cig57 rendered the bacteria significantly compromised for intracellular replication. This intracellular replication defect displayed by the cig57 mutant was complemented upon introduction of cig57 on a plasmid. A yeast-two hybrid screen of a HeLa cell cDNA library identified FCH domain only protein 2 (FCHO2) as a potential eukaryotic binding partner of Cig57. FCHO2 is a membrane binding protein that is involved in the nucleation of clathrin-coated pits. Using the yeast-two hybrid system regions of both Cig57 and FCHO2 that are important for this interaction have been identified. Immunofluorescence microscopy and immunoprecipitation experiments have confirmed the interaction between Cig57 and FCHO2. In addition, siRNA silencing of FCHO2 demonstrates a functional requirement for this protein in CCV development.