The Hippo signaling pathway is essential for regulating the organ size. A cascade of protein kinases, including Hippo and Warts, suppresses organ growth by inhibiting the Yorkie transcriptional coactivator. Actin cytoskeleton plays a role in the regulation of Hippo signaling, but the role of microtubules in Hippo signaling is not well understood. Here we show that Patronin, known as a microtubule minus end protector, negatively regulates Hippo signaling in Drosophila. Patronin knockdown or mutations results in organ size reduction. Patronin RNAi in wing discs leads to downregulation of Yorkie target gene expression. Growth defects by Patronin RNAi are suppressed by reducing the level of Hippo or Warts. Patronin is co-localized with the Kibra-Merlin complex proteins. We also show physical and genetic interaction between Patronin and the hippo upstream regulators. Overexpression of a kinesin family protein Klp10A reduced tissue size and affects Hippo signaling, like Patronin. Taken together, our data suggest that Patronin and Klp10A antagonistically regulate Hippo signaling by changing microtubule minus end dynamics.