The Hippo signaling pathway governs organ size via coordinating cell proliferation and apoptosis, and its dysregulation causes congenital diseases and cancers. The homeostasis of Hippo pathway is achieved through multiple post translational modifications. Through Drosophila genetic screening, we found that miRNAs were also involved in Hippo pathway regulation. Here, we showed that overexpression of miR-7 resulted in small wings, which were neutralized by miR-7-sponge (miR-7-sp) co-expression. Mechanistically, miR-7 inhibited the expression of Hippo pathway target genes. Epistatic analyses revealed that miR-7 modulated Hippo pathway through the transcriptional cofactor Yorkie (Yki). Consistently, overexpression of miR-7 decreased Yki protein. We further found a seed sequence of miR-7 in the yki 3'-UTR region. In addition, we discovered that miR-7 was a transcriptional target of Yki. Thus, a negative feedback loop existed for fine tuning Hippo pathway activity. Taken together, our findings uncovered a novel mechanism by which Yki was silenced by miR-7 for Hippo pathway regulation.