Fruit fly is a sophisticated model animal which possesses an evolutionary conserved innate immune system as represented by the Toll signaling pathway in metazoa. Despite a significant contribution for the identification of mammalian Toll-like receptors, the molecular basis of the intracellular Toll pathway in flies still remains unclear. In this study, we found a suitable cell-line for analyzing the Toll pathway and performed a comparative genome-wide RNAi screening comprised of four distinct screens. Based on the bioinformatic analyses, we identified an E3 ligase gene, Sherpa, as a novel signaling component. Sherpa deficient flies showed susceptibility to bacterial infection as well as the defects in the antimicrobial-peptide production against Gram-positive bacteria and fungi. Cell-based reporter assays indicated that Sherpa functions genetically downstream of Drosophila MyD88 (dMyd88), an adaptor protein for the Toll receptor, and upstream of Tube and Pelle, Drosophila homologues for mammalian IRAK4 and IRAK1/2. Co-immunoprecipitation assays showed that Sherpa interacts with dMyd88 and induces polyubiquitination on dMyd88 and Sherpa itself. Immunofluorescence revealed that Sherpa localizes on the plasma membrane and maintains the proper membrane localization of dMyd88-Tube signaling complex. These results suggested that Sherpa leads adaptor proteins to the plasma membrane around the Toll receptor via polyubiquitinations.