Galectin-3 binding protein (Gal-3BP) is a large hyperglycosylated protein that acts as a ligand for several galectins through glycan-dependent interactions. Gal-3BP can induce galectin-mediated tumor cell aggregation to increase the survival of cancer cells in the bloodstream during the metastatic process. However, the galectin interacting with Gal-3BP and its binding specificity has not been identified and structurally elucidated, mainly due to the limitation of mass spectrometry in glycan sequencing. To understand the role of Gal-3BP, we here used liquid chromatography mass spectrometry combined with specific exoglycosidase reactions to determine the sequences of N-glycans on Gal-3BP from MCF-7 and MDA-MB-231 cells, especially the sequences with terminal sialylation and fucosylation, and addition of LacNAc repeat structures. The N-glycans from both strains are complex type with terminal alpha 2,3-sialidic acid and core fucose linkages, with additional alpha 1,2- and alpha 1,3 fucose linkages found in MCF-7 cells. Compared with that from MCF-7, the Gal-3BP from MDA-MB-231 cells had fewer tetra-antennary structures, only alpha 1,6-linked core fucoses, and more,LacNAc repeat structures; the MDA-MB-231 cells had no surface galectin-3 but used surface galectin-1 for interaction with Gal-3BP to form large oligomers and cell aggregates. This study elucidates the specificity of Gal-3BP interacting with galectin-1 and the role of Gal-3BP in cancer cell aggregation and metastasis.