Large tumor suppressor kinase (LATS2) activity has been shown to be essential to maintain tissue homeostasis by inhibiting the activity of oncoprotein Yes-associated protein (YAP). Many studies have indicated that several new regulators were involved in this process. Citron kinase (CIT) was found to be connected with many components of the Hippo pathway, suggesting a possible function of CIT in regulating the Hippo pathway. However, the molecular mechanism of CIT remains poorly understood, except CIT’s role in cytokinesis. Here, we propose a model in which CIT acts as a scaffold protein linking LATS2 and YAP. Furthermore, our data highlight that CIT binds to and suppresses LATS2 phosphorylation at the hydrophobic motif targeted by MST1. Consequently, LATS2 becomes inactive and YAP is active. Additionally, we found that Sticky, the CIT homolog in Drosophila melanogaster, synergized with Warts to control eye development. Taken together, our study supports a new role of citron kinase as a novel regulator of the Hippo pathway.