In a genome wide screen for interactors of Drosophila Acinus, a regulator of autophagy and endocytic trafficking, we discovered a strong genetic interaction between Acinus and members of the Hippo signaling pathway. For example, loss of one copy of Hippo suppresses the rough eye phenotype produced by GMR-Gal4 over-expression of Acinus. Additionally, over-expression of Acinus suppresses overgrowth caused by over-expression of the gain of function Yorkie-S168A. In order to better understand these genetic interactions, we screened Acinus gain and loss of function for developmental growth phenotypes. Over-expression of Acinus in the wing causes small wings with Engrailed-Gal4, Ms1906-Gal4, or Nubbin-Gal4. Similar to known negative regulators of Yorkie, knock-down of Acinus by two independent RNAi lines using the Engrailed-Gal4, Nubbin-Gal4, or Ms1096-Gal4 drivers causes wing over-growth. Finally, RNAi knock-down of Acinus in the wing causes increased expression of the expanded, four-jointed, and thread (diap1) LacZ reporters, which are used to measure Yorkie transcriptional activity. Taken together, these data suggest that Acinus is a negative regulator of the transcriptional output of the Hippo signaling cascade. A complex, yet poorly understood connection between autophagy and Hippo signaling has been shown in Drosophila; however a clear mechanism to connect these two critical processes remains unknown. Understanding if the connection between Acinus and Hippo signaling is autophagy dependent is a current focus of our research. Moreover, elucidating the mechanism by which Acinus is repressing transcriptional targets of Hippo signaling will provide valuable insight into its function in cellular processes critical for developmental cell growth, as well as cancer.