Inherited deficiency for arylsulfatase (Ars) leads to lysosomal storage of sulfated compounds and to serious diseases such as growth retardation, heart failure, and demyelination in the central nervous system. Ars has been regarded as a lysosomal enzyme because of its hydrolytic activity on synthetic aromatic substrates and the lysosomal localization of its enzymatic activity. We previously demonstrated that a large portion of the mammalian arylsulfatase A (ArsA) protein exists on the cell surface of vascular endothelial cells, suggesting that ArsA plays a role in the components of the extracellular matrix. Here we show that ArsA functions as a substrate on which cells adhere and form protrusions. Coating culture plates with recombinant mouse ArsA (rmArsA) stimulates adhesion of human microvascular endothelial cells to the plate followed by the formation of cell protrusions as well as lamellipodia. rmArsA affects the architecture of the cytoskeleton, with a high density of actin filaments localized to peripheral regions of the cells and the extension of bundles of microtubules into the tips of cellular protrusions. rmArsA also affects the distribution pattern of the cell adhesion-associated proteins, integrin α2β1, and paxillin. rmArsA seems to modulate signaling of basic fibroblast growth factor (bFGF) stimulating cytoskeletal rearrangement. We also show that rmArsA tightly binds to sulfated polysaccharides. We suggest that mammalian ArsA plays a role as a novel component of the extracellular matrix. This viewpoint of Ars could be very useful for clarifying the mechanisms underpinning syndromes caused by the deficiency of the function of Ars genes.