O-fucosylation of Thrombospondin Type 1 Repeats (TSRs) is rare but essential protein modification that occurs on a number of secreted extracellular matrix proteins. O-fucosylation is carried out by two specific enzymes. Protein O-fucosyltransferase 2 (POFUT2) adds a fucose sugar to TSRs and β3-glucosyltransferase (B3GLCT) extends the fucose sugar to create a β3 linked glucose-fucose disaccharide. In order for POFUT2 and B3GLCT to modify TSRs, a consensus sequence, C1-X-X-(S/T)-C2 (Group 1 TSRs) C2-X-X-(S/T)-C3 (Group 2 TSRs), must occur within the TSR. Additionally, POFUT2 and B3GLCT only modify properly folded TSRs, and recent data has shown that O-fucose modification stabilizes and locks TSRs into their proper three-dimensional fold. This recognition of properly folded TSRs and the stabilization that O-fucose suggests that POFUT2 and B3GLCT modification are involved in a non-canonical quality control system for O-fucose substrates containing TSRs. However, the mechanism behind how this stabilization is occurring is not yet known. Therefore, we hypothesized that the O-fucose disaccharide interacts favorably with proximal amino acids. Here we show that the O-fucose disaccharide does in fact interact favorably with amino acids close to its vicinity and also physically protects a conserved disulfide bond by disallowing exposure to the solvent environment. We also demonstrate that upon loss of O-fucosylation, certain O-fucose substrates exhibit loss of secretion in vitro and in vivo. Additionally, we provide a specific circumstance where O-fucosylation is observed to be dispensable for trafficking and secretion through the endoplasmic reticulum and the Golgi apparatus. Together these findings further advance our knowledge of the rare O-fucose modification and potential new functionality of O-fucosylation downstream.