Fibroblast growth factor-2 (Ffg2) is involved in several biological functions. Fgf2 requires glycosaminoglycans, like chondroitin/dermatan sulfate (CS/DS) as co-receptors. CS/DS are linear polysaccharides composed of repeating disaccharide units [-4GlcUAβ1-3-GalNAc-β1-] and [-4IdoUAα1-3-GalNAc-β1-], which can be sulfated. Uronyl 2-O-sulfotransferase (Ust) introduces sulfation at the C2 of IdoUA and GlcUA resulting in over-sulfated units. CHO-K1 cells over-expressing Ust contain significantly more CS/DS 2-O sulfated units, while Ust knock-down abolished CS/DS 2-O sulfation. Structural difference of CS/DS resulted in altered Fgf2 binding and increased p-ERK1/2. As functional consequence of CS/DS 2-O sulfation and altered Fgf2 binding, cell migration and paxillin activation was increased. Inhibition of sulfation, knock-down of Ust and inhibition of FgfR resulted in reduced migration. Similarly, Fgf2 treatment increased migration, which was abolished by Ust knock-down in 3T3 cells. The proteoglycan controlling the CHO migration was syndecan1. Knock-down of Sdc1 in CHO-K1/Ust abolished cell migration. We conclude that the presence of distinctly sulfated CS/DS can tune the Fgf2 effect on cell migration.