Although organelles such as endoplasmic reticulum and the Golgi apparatus are highly compartmentalized, these organelles are interconnected through a network of vesicular trafficking. The marine sponge metabolite ilimaquinone (IQ) is known to induce Golgi membrane fragmentation and is widely used to study the mechanism of vesicular trafficking. Although IQ treatment causes protein kinase D (PKD) activation, detailed mechanism of IQ-induced Golgi membrane fragmentation remains unclear. In the present studies we have found that IQ treatment of cells caused a robust activation of phospholipase D (PLD). In the presence of 1-butanol but not 2-butanol IQ-induced Golgi membrane fragmentation was completely blocked. In addition, IQ failed to induce Golgi membrane fragmentation in PLD-knockout DT40 cells. Furthermore, IQ-induced PKD activation was completely blocked by treatment either with 1-butanol or propranolol. Importantly, IQ-induced Golgi membrane fragmentation was also blocked by propranolol treatment. These results indicate that PLD-catalyzed formation of phosphatidic acid is a prerequisite for IQ-induced Golgi membrane fragmentation and that enzymatic conversion of phosphatidic acid to diacylglycerol is necessary for subsequent activation of PKD and IQ-induced Golgi membrane fragmentation.