Feglymycin (FGM), a natural Streptomyces-derived 13mer peptide, consistently inhibits HIV replication in the lower μM range. FGM also inhibits HIV cell-to-cell transfer between HIV-infected T cells and uninfected CD4(+) T cells and the DC-SIGN-mediated viral transfer to CD4(+) T cells. FGM potently interacts with gp120 (X4 and R5) as determined by SPR analysis and shown to act as a gp120/CD4 binding inhibitor. Alanine-scan analysis showed an important role for l-aspartic acid at position 13 for its anti-HIV activity. In vitro generated FGM-resistant HIV-1 IIIB virus (HIV-1 IIIB(FGMres)) showed two unique mutations in gp120 at positions I153L and K457I. HIV-1 IIIB(FGMres) virus was equally susceptible to other viral binding/adsorption inhibitors with the exception of dextran sulfate (9-fold resistance) and cyclotriazadisulfonamide (>15-fold), two well-described compounds that interfere with HIV entry. In conclusion, FGM is a unique prototype lead peptide with potential for further development of more potent anti-HIV derivatives.