Human papillomaviruses (HPV) are small non-enveloped DNA viruses that can cause a variety of anogenital cancers. During initial infection, HPV16, the best-studied and most prevalent cancer causing type, requires heparan sulfates (HS) for initial binding to host cells. The interaction of HPV16 virions with HS not only enables initial attachment, but also triggers a crucial conformational change in the viral capsid that we termed structural activation. This structural activation facilitates three subsequent alterations of the capsid, which include further conformational but also proteolytic changes in both capsid proteins. These changes are thought to prepare the virus for engaging the elusive internalization receptor, endocytic uptake into host cells and eventual uncoating of the viral genome. Since virus engagement of HS proteoglycans (HSPGs) occurs independently of the protein backbone, we asked, whether the virus would require a specific HS sulfation pattern for the binding and subsequent structural activation of the virus. Employing complimentary approaches using siRNA-mediated knockdown and overexpression of sulfotransferases/sulfatases as well as specifically sulfated/desulfated HS analogues of different length, we found that an HS chain consisting of between 20 and 40 saccharide units was required for stable binding and structural activation. In addition, N- and 6-O-sulfations were needed for HPV infection, whereas only N-sulfation was crucial for the induction of structural activation. Interestingly, 2-O-sulfation and isomerization to iduronic acid appeared to be detrimental for HPV infection. The potential role of 3-O-sulfation is still being investigated. Nevertheless, our work so far already demonstrated that a specific sulfation pattern is required for HPV16 binding and structural activation.