Over the last decades, several studies have reported emissions of nitrous oxide (N2 O) from microalgal cultures and aquatic ecosystems characterized by a high level of algal activity (e.g. eutrophic lakes). As N2 O is a potent greenhouse gas and an ozone-depleting pollutant, these findings suggest that large-scale cultivation of microalgae (and possibly, natural eutrophic ecosystems) could have a significant environmental impact. Using the model unicellular microalga Chlamydomonas reinhardtii, this study was conducted to investigate the molecular basis of microalgal N2 O synthesis. We report that C. reinhardtii supplied with nitrite (NO2- ) under aerobic conditions can reduce NO2- into nitric oxide (NO) using either a mitochondrial cytochrome c oxidase (COX) or a dual enzymatic system of nitrate reductase (NR) and amidoxime-reducing component, and that NO is subsequently reduced into N2 O by the enzyme NO reductase (NOR). Based on experimental evidence and published literature, we hypothesize that when nitrate (NO3- ) is the main Nitrogen source and the intracellular concentration of NO2- is low (i.e. under physiological conditions), microalgal N2 O synthesis involves the reduction of NO3- to NO2- by NR followed by the reduction of NO2- to NO by the dual system involving NR. This microalgal N2 O pathway has broad implications for environmental science and algal biology because the pathway of NO3- assimilation is conserved among microalgae, and because its regulation may involve NO.