A series of trivalent CD4-mimetic miniproteins was synthesized, in which three CD4M9 miniprotein moieties were tethered on a threefold-symmetric scaffold. The trivalent miniproteins were designed to target the CD4-binding sites displayed in the trimeric gp120 complex of HIV-1. The synthesis takes advantage of the highly efficient ligation between a cysteine-tagged CD4M9 miniprotein and a suitable trivalent maleimide that varied in the nature and length of spacer. Antiviral assay revealed that most of the synthetic trivalent miniproteins demonstrated significantly enhanced anti-HIV activities over the monomeric CD4M9 against both R5- and X4-tropic viruses, indicating the beneficial multivalent effects. One compound that possesses a hydrophobic linker was shown to be 140-fold more active than CD4M9 against HIV-1(Bal) infection, implicating a positive contribution of the lipid portion to the antiviral activity. It was also found that most of the trivalent miniproteins showed comparable anti-HIV activities in comparison with a typical bivalent miniprotein, regardless of the length of the linker. The results implicate a novel mechanism of the interactions between the multivalent inhibitors and the trimeric gp120 complex.