Typically found as environmental microorganism, Legionella pneumophila can become accidental pathogen, causing Legionnaires' disease. On inhalation of contaminated aerosols, the bacteria are phagocytised by lung macrophages where they replicate in a specialized compartment – Legionella containing vacuole (LCV). LCV biogenesis is facilitated by the bacterial Dot/Icm Type IV secretion system; which also translocate ~300 bacterial effector proteins into host cells. This secretion of effector proteins occur very rapidly after infection, but only in the presence of host cells. Thus, we hypothesized that active participation by and/or interaction with the host cell is required for the activation of the Dot/Icm system. The aim of this study was to identify these host factors using a high-throughput genome-wide RNAi screen coupled with a FRET-based fluorescence reporter assay for quantifying effector protein translocation to indicate level of activation of the Dot/Icm system. Following extensive optimization, the screening strategy was successfully established in HeLa cells and adapted to robotics at the Victorian Centre for Functional Genomics facility. On completion of the primary screening of the entire human genome, ~520 potential host ‘hits’ were identified that altered the rate of effector protein translocation – leading to either > 30% reduction or < 20% increase in Dot/Icm mediated effector translocation. A more stringent validation screen subsequently confirmed 3 ubiquitin-conjugating (E2s) and 2 ubiquitin ligases (E3s) to be of interest. Upon further replication studies, we found that while these play a role in aiding the translocation of Legionella effector proteins, only a few of them appear to play a part in supporting the bacterial replication. In conclusion, we identified the eukaryotic ubiquitin pathway as important in the pathogenesis of L. pneumophila. This represents the first such methodical, comprehensive study of host factors that influence translocation of effector proteins by Legionella.