The attachment of viruses to the host cell surface is a critical stage that will largely condition cell permissivity and productive infection. The understanding of such mechanisms is therefore essential for gene therapy applications involving viruses, as this step will influence both targeting and delivery efficiency of the gene of interest. Viral attachment depends upon the recognition and binding of viral envelope/capsid proteins to specific cellular receptors that can be from very diverse origins. Amongst them are heparan sulphate proteoglycans (HSPGs), a family of glycoproteins which, through the large binding properties of their heparan sulphate (HS) polysaccharide chains, serve as attachment receptor for a great number of viruses. The aim of this review is to provide an update on the multiple roles of HSPGs during viral infection, with a special focus on viruses used as gene delivery vectors. Consequences of HS binding for gene therapy applications will be assessed, as well as the various strategies that have been developed to potentiate the advantages or to overcome the drawbacks resulting from viral vector interaction with HS.