Summary The neonatal Fc receptor (FcRn) is a major histocompatibility complex class-I related molecule that plays a key role in in several immunological and non-immunological processes, such as transfer of immunoglobulin G (IgG) from mother to child, bidirectional trans epithelial transport of IgG across cellular barriers, antigen presentation by dendritic, and half-life regulation of IgG and albumin. FcRn binds IgG and albumin in a strictly pH-dependent manner, a hallmark which is a prerequisite for all its functions. To fully understand how FcRn mediates its functions, it is necessary to understand how FcRn binds and transports its ligands in different tissues and cell types. N-glycosylation is the most common post-translational modification, and FcRn is N-glycosylated in the extracellular part. The structures of the attached N-glycans, their potential influence on stability, or ligand binding and transport, have not been studied in great detail. Interestingly, one of the major differences between the mouse and human forms of FcRn is that the mouse FcRn (mFcRn) has four N-glycosylation sites in the heavy chain (HC) while human FcRn (hFcRn) has one only. Thus, the aim of this study was to map the profile of N-glycans attached to FcRn across species, and to investigate how the N-glycans affected thermal stability and binding to IgG and albumin. Here, truncated receptors from various species were produced with a glutathione S-transferase (GST) -tag, and expressed by human embryonic kidney (HEK) 293E cells. N-glycan profiles were mapped using mass spectrometry (MS), which revealed distinct and complex profiles. Furthermore, focusing on the mouse and human receptors, we constructed variants with an introduced mutation in the asparagine (N) residue of the N-glycosylation motif to prevent N-glycan attachment. We further addressed how the absence of N-glycans affected thermal stability by using differential scanning fluorimetry (DSF). These experiments showed that the mutations had no or little effect on stability. Finally, we investigated how the FcRn variants bound albumin and IgG. Using an established enzyme linked immunosorbent assay (ELISA), and surface plasmon resonance (SPR) assays, we demonstrated that the mutations had no, or only minor influence on albumin binding, whereas IgG binding was not affected. Specifically, the most interesting finding, in regard to albumin binding, was that mutating the conserved N-glycosylation site within the α2-domain of hFcRn slightly improved the affinity towards human serum albumin (HSA). The same mutation in the mouse FcRn did not modulate binding to mouse serum albumin (MSA). Hence, our data revealed complex N-glycosylation patterns for mouse and human FcRn, and, when the N-glycans were eliminated by mutagenesis, no major effect on stability or ligand binding was observed. Further investigations are needed to address how N-glycosylation of the receptors affect cellular distribution and ligand transport in different cell types.