Immune evasion is a hallmark of the HIV-1 envelope and represents a major barrier to vaccine development. We recently discovered that 2 conserved tyrosines (Y173, Y177) in the V2 loop of the gp120 envelope glycoprotein can be post-translationally modified by O-sulfation and functionally mimic the sulfotyrosines present in the N-terminal region of CCR5, stabilizing the intramolecular interaction between V2 and V31. To gain further insight into the functional role of the V2 sulfotyrosines, we examined the effects of tyrosine sulfation modulation and mutagenesis on HIV-1 neutralization sensitivity. Inhibition of tyrosine sulfation increased HIV-1 sensitivity to soluble CD4 and poorly/non-neutralizing mAbs; at the same time, neutralization by trimer-specific mAbs was reduced, suggesting that tyrosine sulfation contributes to stabilizing the closed trimer conformation. Reciprocal results were obtained upon enhancement of tyrosine sulfation. An even more dramatic effect was observed upon phenylalanine or alanine substitution of the V2 tyrosines, indicating that the tyrosine side-chains play a stabilizing role irrespective of their sulfation status. Strikingly, the V2 tyrosine mutants became highly susceptible to neutralization by HIV-1-infected patient sera, including those with weak/estricted neutralizing capacity, suggesting that the bulk of host-produced antibodies cannot reach through the tight protective shield of the native trimer. Altogether, these results document the key role played by the V2 tyrosines, particularly in their sulfated form, as a mechanism of HIV-1 immune evasion.