Galectin-8 has two carbohydrate recognition domains (CRDs), both of which bind beta-galactosides, but have different fine specificity for larger saccharides. Previously we found that both CRDs were needed for efficient cell surface binding and signaling by soluble galectin-8, but unexpectedly binding of the N-CRD to its best ligands, alpha2-3-sialylated galactosides, was not needed. In search for another role for this fine specificity, we now compared endocytosis of galectin-8 in Chinese hamster ovary (CHO) cells and in a mutant (Lec2) lacking sialylated glycans, by fluorescence microscopy. Galectin-8 was endocytosed in both cells by a non-clathrin and non-cholesterol dependent pathway, but surprisingly, the pathway after endocytosis differed dramatically. In wild type (wt) cells, galectin-8 was found along the plasma membrane, near the nucleus, and in small vesicles. In the Lec2 cells, galectin-8 was found in larger vesicles evenly spread in the cell, but not along the plasma membrane or near the nucleus. A galectin-8 mutant with an N-CRD having reduced affinity to sialylated glycans and increased affinity for other glycans, gave a Lec2 like pattern in the wt CHO cells, but a wt pattern in the Lec2 cells. Moreover, the pattern of galectin-3 after endocytosis differed from that of both the wt and mutant galectin-8. These data clearly demonstrate a role of galectin fine specificity for intracellular targeting.