Exocytosis, by which secretory vesicles are directed to outer cell membrane and subsequently discharge their contents upon triggering, is an important and ubiquitous cellular process responsible for secretion of neurotransmitters from neurons and hormones from endocrine cells. Exocytosis is highly regulated through the orchestrated actions of various proteins and lipids. It has been well recognized that protein kinase Cs (PKCs) are active regulators of exocytosis via phosphorylating multiple key proteins in the exocytotic cascade. The specific actions mediated by PKCs, however, have not been fully elucidated. The challenges mainly lie in the following aspects: 1) large variety of PKC isoforms and dubious specificity of the commonly used pharmacological tools; 2) large variety of PKC substrates; 4) difficulties to isolate the molecular steps in the highly dynamic exocytotic process.