Abstract T20 and maraviroc are the only two currently available entry inhibitors that have shown efficacy in treating HIV-1-infected individuals who have failed to respond to first-line antiretroviral drugs. Gossypol is a polyphenolic aldehyde extracted from cotton plants. By modifying the (−) enantiomer of gossypol with a series of small molecules, we have found that neutral amino acids with aliphatic group derivatives of (−) gossypol show the strongest inhibitory activity and the lowest cytotoxicity in vitro among all the derivatives tested. Additionally, the selectivity index of the (−) gossypol-neutral amino acid conjugates is increased 100-fold when compared with (−) gossypol alone. It is widely accepted that gossypol and gossypol derivatives inhibit HIV-1 replication by targeting reverse transcriptase. However, from the results of our time-of-addition assay, HIV-1-mediated cell fusion assay and VSV-G pseudotyped virus assay, we demonstrate that the alanine-(−) gossypol derivative ((−)G-Ala) is an effective HIV-1 entry inhibitor. Further mechanistic analysis revealed that (−)G-Ala neither blocks gp120-CD4 binding nor interacts with the HIV-1 co-receptor CXCR4. Results from sandwich ELISA, native-PAGE and circular dichroism (CD) show that (−)G-Ala inhibits the cell fusion-activated gp41 core domain. Moreover, (−)G-Ala binds to the HIV-5-Helix protein and blocking D-peptide (PIE7) binding to the hydrophobic pocket on the surface of the gp41 internal trimeric coiled-coil domain. The contraceptive properties of (−) gossypol and amino acid derivatives of (−) gossypol are also discussed. Collectively, our results indicate that (−)G-Ala may bind to the gp41 hydrophobic pocket and block the formation of the cell fusion-activated gp41 core to inhibit HIV-1-mediated membrane fusion and subsequent viral entry.