Heparan sulfate as an attachment factor for ASFV is restricted by host HB-EGF

African swine fever virus (ASFV) is a highly virulent pathogen that poses a significant threat to the global pig industry, and no vaccines or effective treatments are currently available. Cellular attachment is initial and crucial for viral entry and subsequent infection. Understanding the molecular mechanisms of ASFV entry is therefore essential for developing antiviral strategies. Here, we show that cellular heparan sulfate (HS) assists ASFV attachment and subsequent entry into the target cell. Chemical inhibitors of HS and competition assays using heparin and heparan sulfate impair ASFV attachment and infection in the cultured cells. The direct binding between the ASFV virion and heparin is demonstrated by using heparin-coated beads, and this interaction can be blocked by free heparin. In addition, we identify host HB-EGF as a restriction factor against ASFV and reveal that the heparin-binding domain (HBD) is critical for its antiviral activity. The HBD-derived peptide exhibits strong binding affinity for heparin and exerts potent antiviral activity against ASFV. Together, our findings indicate that HS acts as an attachment factor for ASFV, and pharmacologically targeting it may represent a potential strategy for antiviral development.