Extracellular matrix (ECM) composition and structural integrity is one of many factors that influence cellular differentiation. Fibronectin (FN) which is in many tissues the most abundant ECM protein forms a unique fibrillary network. FN homes several binding sites for sulfated glycosaminoglycans (sGAG), such as heparin (Hep), which was previously shown to influence FN conformation and protein binding. Synthetically sulfated hyaluronan derivatives (sHA) can serve as model molecules with a well characterized sulfation pattern to study sGAG-FN interaction. Here is shown that the low-sulfated sHA (sHA1) interacts with FN and influences fibril assembly. The interaction of FN fibrils with sHA1 and Hep, but not with non-sulfated HA was visualized by immunofluorescent co-staining. FRET analysis of FN confirmed the presence of more extended fibrils in human bone marrow stromal cells (hBMSC)-derived ECM in response to sHA1 and Hep. Although both sHA1 and Hep affected FN conformation, exclusively sHA1 increased FN protein level and led to thinner fibrils. Further, only sHA1 had a pro-osteogenic effect and enhanced the activity of tissue non-specific alkaline phosphatase. We hypothesize that the sHA1-triggered change in FN assembly influences the entire ECM network and could be the underlying mechanism for the pro-osteogenic effect of sHA1 on hBMSC.