Interestingly, despite close similarities in the overall tetrameric architecture and identical monovalent CRD-mannose binding motifs, DC-SIGN/R actually display distinct virus binding and trans-infection properties. For example, DC-SIGN is more effective in transmitting the HIV infection than DC-SIGNR, while only DC-SIGNR, but not DC-SIGN, can transmit the West Nile Virus infection. The structural mechanisms underlying such differences remain not fully understood. By developing a new glycannanoparticle based multifunctional probe, we have discovered that DC-SIGN/R clearly exhibit distinct modes and affinities in binding to glycan-nanoparticles, due to subtle differences in their binding site orientation. All four binding sites in DC-SIGN point upwardly in the same direction, but those in DC-SIGNR are split into two pairs and point side-ways. As a result, DC-SIGN binds tetravalently to a single glycan-nanoparticle, whereas DC-SIGNR binds bisdivalently with two different glycan-nanoparticles, resulting in the former binding being significantly stronger than the latter (by ∼20-200 fold). In this paper, we have systematically investigated the pH-dependency of DC-SIGN/R MLGI properties with densely glycosylated gold nanoparticles (glycan-GNPs) over a pH range of 4.6 to 7.4, mimicking the pH range experienced by viruses during the natural cellular uptake and trafficking pathways through endocytosis. We have studied their relative MLGI affinities as a function of pH by exploiting GNP's strong fluorescence quenching properties, and monitored the hydrodynamic diameters (Dhs) of the resulting lectin-glycan-GNP complexes during this process, revealing that the MLGIs of DC-SIGN/R display distinct pH dependency. We have further investigated the reversibility of DC-SIGN/Rglycan-GNP binding by cycling pH between 4.6 and 7.4, showing the process is reversible. Our results thus provide a useful insight into the pH-dependent glycan binding and release properties for these important lectin viral receptors.