N-glycosylation is a major post-translational modification of proteins that has a crucial influence on cell targeting, activity, and half-life. This process starts in the endoplasmic reticulum, where an oligosaccharide precursor is added to the newly synthesized protein, and continues in the Golgi apparatus, where the N-linked carbohydrate sequences are processed. Importantly, most approved recombinant pharmaceutical proteins (so-called biologics) are glycoproteins mainly produced in mammalian cells, which is a lengthy, costly, and complex process. Today, several microalgae such as the diatom Phaeodactylum tricornutum, and the green microalgae Chlamydomonas reinhardtii, Chlorella vulgaris, and Dunaliella salina are considered efficient and eco-friendly alternative platforms for the production of biologics. However, unlike C. reinhardtii, C. vulgaris, and P. tricornutum, there is to date no data reported regarding the protein N-glycosylation pathway in D. salina. Here, we first investigated the protein N-glycosylation in this green microalga by MALDI-TOF mass spectrometry. These analyses showed that proteins from D. salina are N-glycosylated with Man₅GlcNAc₂ oligomannoside. Using genome mining approaches, we then identified genes encoding proteins involved in the N-glycosylation pathways in D. salina. Genetic similarities and phylogenetic relationships of the putative sequences with homologues from C. reinhardtii, P. tricornutum, and humans were investigated. These data revealed that in D. salina, the biosynthesis of nucleotide sugars and N-glycan biosynthesis share mainly similarities with the GnT I-independent pathway of C. reinhardtii, which gives rise to the synthesis of a non-canonical oligomannoside Man₅GlcNAc₂. Although an α(1,3)-fucosyltransferase is identified in the D. salina genome, impairment of the cytosolic GDP-Fuc biosynthesis prevents the Golgi fucosylation of N-glycans. Taken together, these data demonstrated that proteins from D. salina are homogeneously N-glycosylated with a non-canonical Man₅GlcNAc₂.