Nitric oxide (NO) functions as a signaling molecule in many biological processes in green algae and higher plants. Although the mechanisms of NO synthesis in most plants are the subject of ongoing research and debate, a functional NO synthase (NOS) has been characterized only in Ostreococcus tauri. To date, the question of whether NO synthesis occurs in other NOS-containing members of the class Mamiellophyceaea, which gave rise to the core Chlorophyta, has not been elucidated. We found that, like O. tauri, O. lucimarinus and Bathycoccus prasinos grow on arginine as the sole nitrogen source, and their NOSs function and produce NO in cells. Moreover, in O. tauri, O. lucimarinus, and B. prasinos, NO exerts its biological functions through protein S-nitrosylation. Collectively, our data suggest that both NO and S-nitrosylated proteins are important mediators in the process of cell growth in NOS-containing representatives of Mamiellophyceae. Thus, we have updated the data related to protein S-nitrosylation as an evolutionarily conserved mechanism regulating many aspects of cell signaling in plants.