T20 (generic name: Enfuvirtide, brand name: Fuzeon) is the only FDA-approved HIV fusion inhibitor that is being used for treatment of HIV/AIDS patients who have failed to respond to current antiretroviral drugs. However, it rapidly induces drug resistance in vitro and in vivo. On the basis of the structural and functional information of anti-HIV peptides from a previous study, we designed an HIV fusion inhibitor named CP32M, a 32-mer synthetic peptide that is highly effective in inhibiting infection by a wide range of primary HIV-1 isolates from multiple genotypes with R5- or dual-tropic (R5X4) phenotype, including a group O virus (BCF02) that is resistant to T20 and C34 (another anti-HIV peptide). Strikingly, CP32M is exceptionally potent (at low picomolar level) against infection by a panel of HIV-1 mutants highly resistant to T20 and C34. These findings suggest that CP32M can be further developed as an antiviral therapeutic against multidrug resistant HIV-1.