Abstract: The HIV-1 capsid protein (CA) is an effective target for antiviral drug discovery. Targeting the capsid assembly inhibitor peptide (CAI) binding pocket in the C-terminal domain of HIV-1 CA (CA CTD) can be used to screen for CA protein inhibitors. In this study, we found that the small molecule suramin targets HIV-1 CA by binding to this pocket, with an IC₅₀ value of 2.1 μmol·L^-1 for inhibiting the CA CTD-CAI interaction, based on homogeneous time-resolved fluorescence (HTRF) technology. Bio-layer interferometry (BLI) experiments demonstrated that suramin binds directly to CA, with a binding affinity to CA hexamer (K_D = 248 nmol·L^-1) higher than to CA monomer (K_D = 227 μmol·L^-1). In addition, in vitro CA protein assembly assays showed that suramin promotes disordered multimerization of CA protein. Binding model analysis revealed that suramin binds to the interface between adjacent CA monomers in the CA hexamer through the N57, Q63, Q67 and Y169 residues, thereby enhancing CA multimerization. In summary, suramin is a CA protein inhibitor that targets the CA hexamer and can serve as a starting point for the discovery of new CA inhibitors.