Fission yeast its3(+) encodes an essential phosphatidylinositol-4-phosphate 5-kinase (PI4P5K) that regulates cell integrity and cytokinesis. We performed a genetic screen to identify genes that function in PI4P5K-mediated signaling, and identified gyp10(+) encoding a Rab GTPase-activating protein (GAP), a negative regulator for Rab GTPase signaling. Its3 overproduction caused growth defects and abnormal cytoplasmic accumulation of the Its3 protein, which can be stained by calcofluor. Notably, Its3 overproducing cells displayed abnormal membranous structures, multilamella Golgi and fragmented vacuoles showed by Electron microscopy. Furthermore, the excess cytoplasmic Its3 structure partly colocalized with the fluorescence of FM4-64. Gyp10 rescued both growth defects and abnormal Its3 localization when it was over-expressed. Gyp10 functionally interacted with the Rab GTPases Ypt3 and Ryh1, both of which regulate Golgi membrane trafficking. Consistently, mutation or deletion of Ypt3 and Ryh1 suppressed phenotypes associated with Its3 overproduction. Importantly, the plasma membrane localization of Its3 was also affected by the impairment of the Ypt3/Ryh1 Rab membrane trafficking, thus suggesting that membrane trafficking events regulated by two Rab GTPases functionally interacts with PI4,5P(2) signaling. These results suggest a mechanism whereby PI4P5K signaling/localization is affected by Golgi membrane trafficking, thus provide a functional link between the PI4,5P(2) signaling and Rab-mediated trafficking.