The Hippo pathway regulates growth and apoptosis. We identify RhoBTB proteins as novel regulators of Hippo signaling. RhoBTB depletion in the Drosophila wing disc epithelium cooperated with Yki to drive hyperplasia into neoplasia. Depletion of RhoBTB2 caused elevated YAP activity in human cells. RhoBTB2 deficiency resulted in increased colony formation in assays for anchorage-independent growth. We provide evidence that RhoBTBs acts on Hippo signaling through regulation of the kinase LKB1. LKB1 protein levels were reduced upon RhoBTB2 depletion, which correlated with increased LKB1 ubiquitination. Restoring LKB1 levels rescued loss of RhoBTB in Drosophila Our results suggest that RhoBTB-dependent LKB1 regulation may contribute to its tumor-suppressive function.