Fibrillin-1 (FBN1) is an essential component of the extracellular matrix, forming microfibril bundles that are important for proper development of elastic tissues found in the aorta and lung, as well as non-elastic tissue found in the eyes and skeleton. Many missense mutations in the FBN1 gene are associated with Marfan syndrome (MFS), a common developmental disorder. FBN1 contains 47 Epidermal Growth Factor-Like (EGF) repeats, which are protein domains characterized by six cysteines and three disulfide bonds. Over half of these EGF repeats are modified with an O-glucose monosaccharide added by Protein O-glucosyltransferase 2 and/or 3 (POGLUT2/3). Previous studies showed that O-glucose modifies the serine within the putative consensus sequence between cysteines 3 and 4: C3-x-N-T-x-G-S-F/Y-x-C4. These residues are common among modified EGFs, but it is unknown if they are required for O-glucosylation. To address this, we used a glycoproteomic approach by analyzing O-glucosylation levels of individual EGF repeats from overexpressed N-terminal FBN1 variants in HEK293T cells. Surprisingly, only the serine (S) was required for O-glucosylation, leading to the revised consensus sequence, C3-x-x-x-x-x-S-x-x-C4. Using this open consensus in database searches, the possible number of POGLUT2/3 substrates in humans has doubled. While some variants displayed reduced O-glucose monosaccharide modification, other variants, including MFS variants, displayed elongation of the O-glucose monosaccharide by additional glycosyltransferases. MFS variants reduction or elongation of O-glucose warrants further investigation on their influence on FBN1 function, which could play a role in the molecular mechanism of the disease.