While the variations in the HIV-1 Env V3 loop have been the focus of much research to explore its functional effect, how specific mutations of certain amino acids in the V3 loop affect viral fitness remains unclear. In this study, we evaluated a series of natural polymorphisms at positions 328 and 330 with different combinations of adjacent glycosylation sites in the HIV-1 subtype B backbone to address their role in replicative fitness and sensitivity to entry inhibitors based on analysis of env sequence frequency at the population level. Pairwise growth competition experiment showed that wild-type virus with major consensus amino acids obviously had higher replicative fitness (P < 0.001). A change at position 328 to a less frequently occurring amino acid, K, together with a mutated N332 glycosylation site harbored lower fitness and became more sensitive to coreceptor antagonists (AMD3100), fusion inhibitors (T20) and sCD4. A change at position 330 to a less frequently occurring amino acid, Y, together with a mutated N332 glycosylation site resulted in higher fitness and less sensitivity to entry inhibitors (T20, AMD3100, and sCD4), and viruses containing both changes showed intermediate activity. It seems that the H330Y mutation compensated for the reduced replicative capacity caused by the Q328 K mutation. Moreover, viruses that showed lower replicative fitness also exhibited slower entry kinetics, lower levels of replication intermediates and protein packaging, and a lower ability to replicate in primary peripheral blood mononuclear cells (PBMCs). The findings highlight the functional effect of variations at 328 and 330 in the V3 loop on replicative fitness and may benefit HIV-1 treatment by helping predict the sensitivity to entry inhibitors. © 2021