Many proteins can form biomolecular condensates in cells through liquid-liquid phase separation (LLPS). LLPS elucidates many membrane-less organelle events in recent years. In our previous study, we reported monomeric galectin-3 undergoes LLPS to form oligomers through aromatics residues on its disordered N-terminal domain (NTD). Galectin-3’s function is agglutinating glycosylated molecules in many biological functions such as lysosome repairing, autophagy, and immunity. The LLPS mechanism of galectin-3 is contributed by NTD to NTD and NTD to CRD (carbohydrate recognition domain) self-association [1]. However, the detailed molecular mechanism of NTD to CRD remains unclear. Our recent isothermal titration calorimetry (ITC) and tryptophan fluorescence spectrum (TFS) data reveal that the F face (NTD-binding face of CRD) binding events have no significant effect on S face (Sugar-binding face of CRD) sugar-binding ability. It shows that self-association mainly regulates the galectin-3’s agglutination but not sugar recognition. Next, we used NMR HSQC and T2 experiments to identify the molecular property of galectin-3 variants (point mutations are based on sequence alignment of the galectin family and evolution) in different protein concentrations. By comparing the chemical shift of specific residues and molecular tumbling rate, our data shows NTD to CRD self-association of galectin-3 is mainly contributed by cation-π interaction. In addition, the lipopolysaccharide (LPS) micelle model also shows that agglutinated function is interrupted when NTD to CRD interactions are abolished. Combined with our previous study, our proposed molecular model elaborates the LLPS molecular mechanism of galectin-3 and offers potential and novel drug-targeting of galectin-3.