The controlled regulation of nitric oxide (NO) synthesis in endothelial cells and cardiomyocytes by the endothelial form of nitric oxide synthase (eNOS or NOS3) is essential for cardiovascular health. In recent years, a picture of complex and precise regulation of eNOS activity involving multi-site phosphorylation of specific serine and threonine residues has emerged. Regulation of endothelial NO synthesis by multi-site eNOS phosphorylation occurs in response to a wide variety of humoral, mechanical and pharmacological stimuli. This regulation involves numerous kinases and phosphatases, as well as interactions with other aspects of eNOS regulation such as Ca²⁺ flux, protein–protein interactions and regulation of subcellular localization. Phosphorylation of eNOS-Ser¹¹⁷⁷ close to the carboxy-terminal is a critical requirement for eNOS activation. In addition, phosphorylation of eNOS-Ser⁶³³ in the flavin mononucleotide (FMN) binding domain also increases eNOS activity and appears particularly important for the maintenance of NO synthesis after initial activation by Ca²⁺ flux and Ser¹¹⁷⁷ phosphorylation. In contrast, NO synthesis is inhibited by phosphorylation of eNOS-Thr⁴⁹⁵, which interferes with the binding of calmodulin to the eNOS calmodulin-binding domain. Regulated phosphorylation of eNOS also occurs at eNOS-Ser¹¹⁴ and eNOS-Ser⁶¹⁵; however, the functions of these phosphorylation sites remain controversial. This review summarizes the present knowledge of the regulation of NO synthesis by multi-site eNOS phosphorylation and its relationship to other mechanisms of eNOS regulation. This progress in understanding important mechanisms controlling endothelial NO synthesis creates new opportunities to understand and potentially treat cardiovascular diseases characterized by deficient NO synthesis.