Galectin-9, a member of galectin family, plays multiple roles in a variety of cellular functions, including cell adhesion, aggregation, and apoptosis. Galectin-9 also has three isoforms (named galectin-9L, galectin-9M, and galectin-9S), but whether these isoforms differ in their functions remains poorly understood. In this study, we showed that transient expression of galectin-9L decreased E-selectin levels, while transient expression of galectin-9M or galectin-9S increased E-selectin levels in LoVo cells, which do not express endogenous galectin-9. We also found that over-expression of three galectin-9 isoforms led to increased attachment of LoVo cells to extracellular matrix proteins respectively, while over-expression of galectin-9M or galectin-9S increased the adhesion of LoVo cells to human umbilical vein endothelial cells in vitro. In summary, these findings indicate that different isoforms of galectin-9 exhibit distinct biological functions.