Over the last two decades, the role of NO in cellular processes and signaling has been extensively investigated. Under physiological and pathophysiological conditions, NO is produced primarily by specific NO synthase enzymes and also through the process of nitrite reduction. NO is an important biological signaling molecule in the vasculature and tissues. NO contributes to hypoxic signaling, vasodilation, regulation of intracellular oxygen availability, and cytoprotection. In cells, NO interacts with various target proteins and organelles such as mitochondria and endoplasmic reticulum. Its interaction with intracellular proteins also results in the formation of second messenger molecules such as cyclic guanosine monophosphate. Post translational modification of proteins by NO may be involved in regulating cellular functions and the pathogenesis of human diseases. In NO-mediated intercellular communication, various cellular molecules are involved in its storage and transport. Production of NO by effector cells and the process of its conversion to form reactive nitrogen species modulates the balance of its in vivo actions in cytoprotection and cytotoxicity. Therefore, understanding the cellular signaling processes and biological effects of NO in health and disease can facilitate the development of improved therapeutic approaches to prevent or ameliorate disease. This mini review summarizes the processes of NO formation and NO-mediated cellular signaling and their role in physiology and disease.