Endothelial nitric oxide synthase (eNOS) is a major enzyme responsible for nitric oxide (NO) production. Both eNOS and NO play an important role in regulating vessel structure and function through participating in the physiological and pathological process. Generally, the phosphorylation of eNOS has been recognized as a common mechanism for regulation of eNOS function. However, recent studies have demonstrated several other new mechanisms, such as acetylation, S-glutathionylation, and protein-protein interaction. This review highlights the basic structure and functions of eNOS as well as the relationship between regulation of eNOS activity and cardiovascular diseases.