Enfuvirtide (T20) is the first U.S. FDA-approved HIV fusion inhibitor-based anti-HIV drug. Its clinical application is limited because of its low potency and short half-life. We previously reported that peptide HP23-E6-IDL, containing both N- and C-terminal anchor-tails, exhibited stronger potency and a better resistance profile than T20. Here we designed an analogous peptide, YIK, by introducing a mutation, T639I, and then a lipopeptide, YIK-C16, by adding palmitic acid (C16) at the C-terminus of YIK. We found that YIK-C16 was 4.4- and 3.6-fold more potent than HP23-E6-IDL and YIK against HIV-1IIIB infection and 13.3- and 10.5-fold more effective than HP23-E6-IDL and YIK against HIV-1Bal infection, respectively. Consistently, the ex vivo anti-HIV-1IIIB activity, as determined by the highest dilution-fold of the serum causing 50% inhibition of HIV-1 infection, of YIK-C16 in the sera of pretreated mice was remarkably higher than that of YIK or HP23-E6-IDL. The serum half-life (t1/2 = 5.9 h) of YIK-C16 was also significantly longer than that of YIK (t1/2 = 1.3 h) and HP23-E6-IDL (t1/2 = 1.0 h). These results suggest that the lipopeptide YIK-C16 shows promise for further development as a new anti-HIV drug with improved anti-HIV-1 activity and a prolonged half-life.