In conclusion, we have probed the pH-dependent MLGI properties between DC-SIGN/R and Gx-DiMan via fluorescence quenching and hydrodynamic size studies for the first time. We have revealed that both DC-SIGN/R binding with Gx-DiMan are strongly pH-dependent and partially reversible. Our pHdependent fluorescence quenching studies show that DC-SIGN binds strongly and stably with Gx-DiMan from neutral to weakly acidic pH (e.g., 7.4 to 5.4, comparable to normal physiological to endosomal pH range), but its binding is significantly reduced at lower pH. This correlates well to DC-SIGN's biological function as an endocytic and recycling receptor, which requires it to bind and endocytose target ligands at normal physiological pH, and subsequently release them under the acidic environment of intracellular endosomes for receptor recycling. In contrast, DC-SIGNR exhibits the strongest binding (highest QE) with GNP-DiMan at pH 5.4, and any deviation from this pH leads to progressively reduced affinity (lower QE). This result suggests that DC-SIGNR bound ligands may not be released in early endosomes, instead, they are more likely to be released in the more acidic late endosomes to lysosomes. Our DLS studies have revealed that DC-SIGN/R adopt their characteristic binding modes at pH 7.4 (i.e. tetravalent binding with one single Gx-DiMan for DC-SIGN, crosslinking with different Gx-DiMans for DC-SIGNR), but their binding modes are gradually shifted to crosslinking as pH is further reduced, leading to the formation of larger DC-SIGN/R-Gx-DiMan complexes. We have further revealed that both DC-SIGN/R bindings with GNP-DiMan are partially reversible in a pHdependent manner. Overall, DC-SIGN ligand binding is weakened at weakly acidic pH, but the affinity is reinstalled as pH is switched back to 7.4, consistent with DC-SIGN's role as a recycling endocytic receptor. In addition, DC-SIGNR's strong affinity for Gx-DiMan over pH 5.4-5.8 indicates that it does not release ligands in earlier endosomal pH, making it difficult to act as a ligand recycling receptor. This work thus reveals a new molecular insight into DC-SIGN/R's distinct pH-dependent MLGI behaviours which are fundamental for their biological functions.