Heparan sulfate is found on the surface of most cell types, as well as in basement membranes and extracellular matrices. Its strong anionic properties and highly variable structure enable this glycosaminoglycan to provide binding sites for numerous protein ligands, including many soluble mediators of the immune system, and may promote or inhibit their activity. The formation of ligand binding sites on heparan sulfate (HS) occurs in a tissue- and context-specific fashion through the action of several families of enzymes, most of which have multiple isoforms with subtly different specificities. Changes in the expression levels of these biosynthetic enzymes occur in response to inflammatory stimuli, resulting in structurally different HS and acquisition or loss of binding sites for immune mediators. In this review, we discuss the multiple roles for HS in regulating immune responses, and the evidence for inflammation-associated changes to HS structure.