Pseudomonas aeruginosa is an important opportunistic human pathogen that interacts with phylogenetically diverse nonmammalian hosts, including plants, nematodes, and insects. Here, we exploited the P. aeruginosa-induced killing of the fruit fly Drosophila melanogaster as an assay system to screen for virulence-attenuated mutants of P. aeruginosa PA14. Fifteen nonredundant mutants were isolated from 4,018 random transposon (TnphoA) insertion clones, and 13 out of them (86.7%) displayed significantly reduced virulence in a murine peritonitis model as well. The TnphoA insertion sites of the 15 mutants were determined; already known virulence genes (dsbA, pvdI, fhlB, pilF, and wspF) and new virulence genes such as PA0253 (hudR), PA0369, PA2077, PA0272, PA2113, PA2965 (fabF1), and PA2002 were identified; one insertion was located at the intergenic region between PA1928 and PA1929; and the other two insertions were located in the genes (PA14_35740 and PA14_36000) within a putative genomic island, indicating a potential pathogenicity island of PA14. Further characterization of hudR, a virulence gene which encodes a MarR/SlyA family transcription factor, revealed that elevated expression of PA0254 (hudA [homologous to UbiD]) was necessary and sufficient for the virulence attenuation of the hudR mutant. The HudR protein repressed the hudAR operon by directly binding to its upstream promoter region. Collectively, these results validate the relevance of the D. melanogaster model for the high-throughput identification of new virulence factors involved in the multihost pathogenesis of P. aeruginosa.