SMAD ubiquitination regulatory factors 1 and 2 (Smurf1/2) are members of the HECT domain E3 ligase family which play crucial roles in the regulation of cell cycle progression, planar cell polarity, cancer metastasis and cell apoptosis. We recently showed that the Drosophila homolog dSmurf controls the stability of Warts kinase to regulate the Hippo pathway. In the current study, we found that the F-box protein Slimb controls dSmurf protein level to regulate the Hippo pathway. Slimb physically associates with dSmurf as revealed by co-immunoprecipitation assay in S2 cells. The C-terminal WD40 repeats of Slimb (188-510 amino acid) and the C-terminal HECT domain of dSmurf (723-1061 amino acid) are necessary for their binding. Interaction with Slimb leads to the ubiquitination and degradation of dSmurf, resulting in negative regulation of dSmurf-mediated Yki phosphorylation and activity in the Hippo pathway. Thus our study revealed a new regulatory mechanism of the Hippo pathway which may provide implications for developing tumor treatment. (C) 2016 Elsevier Inc. All rights reserved.