Objective:Screening for HECT E3 ligases that can regulate Sonic Hedgehog (Shh) signaling pathway. Methods:In the first round of screening,siRNAs of the HECT E3 ligases were tested for Shh pathway transduction by GliBS-luciferase assay. And in the second round,these siRNAs were tested for the localization of Patched1-GFP in primary cilia by immunofluorescence staining. Results:After two rounds of screening,5 HECT E3 ligases were identified to regulate Shh pathway,which are Smurf1,Smurf2,Ube3c,Wwp1 and Wwp2. Conclusion:A screening system of new regulators of Shh signaling was setup,and 5 HECT E3 ligases were found from our preliminary screening.