Galectin-3 is a β-galactoside-specific lectin implicated in diverse processes involved in cellular interactions. Recently, the Mac-2-binding protein, a heavily N-glycosylated secreted protein with a subunit M_r of 97,000, was identified as its ligand. The present study characterizes the interaction between galectin-3 and Mac-2-binding protein in whole cells and measures their relative expression levels. Incubation of A375 cells with affinity-purified Mac-2-binding protein resulted in its binding to galectin-3 on the cell surface in a specific carbohydrate-dependent manner. Mac-2-binding protein also induced homotypic cell aggregation, which was inhibited by lactose or Fab' fragments of an anti-galectin-3 antibody. Northern blotting analysis revealed differences in the transcriptional regulation of galectin-3 and Mac-2-binding protein. These results provide the first direct evidence for a Mac-2-binding protein function and suggest that it may play a role in tumor cell embolization during metastasis through interaction with galectin-3.