Background N-glycosylation is a modification of proteins in which oligosaccharide structures are added to protein backbones. Glycans are one of the main regulators of different immune system features and show biomarker potential in diabetes. In our work on children with type 1 diabetes (T1D), we identified a novel genetic regulator of plasma protein N-glycosylation, C3 gene. Complement component 3 (C3) is a central protein of all complement activation pathways. To study the N-glycosylation of C3 we developed a novel high-throughput method. Aims We analyzed C3 glycopeptides to a) compare N-glycans between children with T1D and their healthy siblings, b) search for connections between N-glycans and T1D-related complications in adults, c) define the C3 Nglycosylation in the general population and reveal its genetic regulation. Methods Glycoproteins were enriched from plasma using ConA Sepharose and digested with Glu-C. Glycopeptides were enriched with HILIC silica beads, analyzed by UPLC-MS, and quantified. C3 glycopeptides were analyzed in a) 61 children newly diagnosed with T1D and 84 unaffected siblings from DanDiabKids, b) 189 adults with T1D from Croatia, and c) 816 adults from the general Croatian population (GWAS on glycans). Results We showed that C3 has two N-glycosylation sites, with three high-mannose glycans on Asn63 and six on Asn917. Significant changes were found between children with T1D and healthy siblings, expressed through an increase in glycans with more mannose units, especially the Man9Glc. A regression model showed discriminative power with an AUC of 0.879. Significant changes were also seen in severe albuminuria, and in subjects with hypertension, but less with non-proliferative retinopathy. Glycans were associated with HbA1c levels. Smoking, eGFR, and the disease duration did not affect C3 N-glycome. We also identified 6 genome-wide significant loci associated with C3 N-glycosylation. Conclusions Our results suggest the involvement of C3 N-glycosylation in T1D development and its significant potential for assessment of T1D risk in children. It also has a value in distinguishing subjects with diabetic complications, and the potential to monitor the disease progression. Some gene variants that regulate C3 N-glycans were previously associated with different immune diseases and the activity of the complement activation pathway. Results suggest that C3 glycans could be novel targets in unraveling the pathogenesis of T1D.