The alterations of cell surface sialylation play a key role in tumor metastasis. Enhanced α2,6-sialylation on N-glycans results from overexpression of the sialyltransferase ST6Gal-I. Hca-F and Hca-P cells are murine hepatocarcinoma cell lines which metastasize only to the lymph nodes. Our previous study revealed that ST6Gal-I was involved in the adhesion of Hca-F cells to fibronectin. However, the roles of sialic acids in the adhesion of Hca-F cells to lymph nodes still remain poorly understood. In this study, we observed that expression levels of α2,6-linked sialic acids on Hca-F cells were higher compared to Hca-P cells. Knockdown of ST6Gal-I by small hairpin RNA (shRNA) transfection decreased the expression of α2,6-linked sialic acids and inhibited the adhesion of Hca-F cells to lymph nodes. The adhesion ability was reported to be mediated by siglec-2 which preferentially binds to α2,6-linked sialic acids, and in addition, ST6Gal-I knockdown inhibited the phosphorylated levels of focal adhesion kinase (FAK) and paxillin when cells were treated with siglec-2. Taken together, these results suggest that interaction of α2,6-linked sialic acids with siglec-2 might modulate the adhesion of hepatocarcinoma cells to lymph nodes through the FAK signaling pathway.