Protein kinase D (PKD) is a cytosolic serine-threonine kinase that binds to the trans-Golgi network (TGN) and regulates the fission of transport carriers specifically destined to the cell surface. PKD was found to bind diacylglycerol (DAG), and this binding was necessary for its recruitment to the TGN. Reducing cellular levels of DAG inhibited PKD recruitment and blocked protein transport from the TGN to the cell surface. Thus, a DAG-dependent, PKD-mediated signaling regulates the formation of transport carriers from the TGN in mammalian cells.