Nitric oxide (NO) is an important cellular signaling molecule regulating various physiological processes, in both animals and plants. In animals, NO synthesis is mainly catalyzed by NO synthase (NOS) enzymes. In plants, NOS-like activities sensitive to mammalian NOS inhibitors have been measured, although no sequences encoding mammalian NOSs have been found in land plants. Interestingly, we identified NOS-like sequences in 20 algae species. These latter include the filamentous charophyte green algae Klebsormidium nitens, a biological model to study the early transition step from aquatic algae to land plants. In order to understand the mechanisms governing NO synthesis and signaling in green lineage we initiated the functional characterization of K. nitens NOSs (KnNOS) by analyzing their primary sequences as well as their expression levels in response to abiotic stresses. Currently, two NOSs were identified in K. nitens genome: the KnNOS1 which possesses classical mammalian NOS architecture consisting of oxygenase and reductase domains with some specificities as lack of conserved residues in binding domain of BH4 cofactors; and the KnNOS2 displaying a large C-ter extension containing an ANK motif and a globin domain. The two KnNOSs seem to be regulated in different ways. KnNOS1 exhibited constitutive expression during the conditions tested, whereas KnNOS2 appeared to be transcriptionally regulated during stress. In parallel studies, we also built the in silico protein–protein interaction network of human NOSs using the BioGRID database and human NOS interaction data. Interestingly, genes encoding orthologs of several of these candidates were found in K. nitens genome. Some of these conserved partners are known to be involved in mammalian NOSs regulation and represent interesting candidates for further investigation. Overall these findings open the way for a deeper characterization of KnNOSs and its protein partners and will facilitate further investigation of NO signaling in green lineage.