MicroRNAs (miRNAs) are endogenous non-coding regulatory RNAs involved in multiple cellular processes. Emerging evidences showed that miRNAs are involved in changing the cell surface glycosylation modification and oncogenesis. In this study, the role of miRNA-24-1 in O-GlcNAcylation and metastasis of mouse hepatocarcinoma cells was investigated. miRNAs expression array profiles were obtained from mouse hepatocarcinoma cell lines Hca-P and Hca-F with the low/high lymphatic metastasis potential, respectively. Based on the miRNAs expression array profiles, miRNA-24-1 expression was found to exhibit converse coincidence with metastasis potential, O-GlcNAc transferase (OGT) expression and O-GlcNAcylation. Dual-luciferase reporter assay revealed that miRNA-24-1 specifically binds to 3′-UTR of OGT. Furthermore, transfecting mouse hepatocarcinoma cells with miR-24-1 mimic and antisense oligonucleotide showed miR-24-mediates OGT expression silencing. This silencing is associated with the suppression of cell metastasis potential, down-regulation of the O-GlcNAcylation on c-Myc and decrease of c-Myc expression at the protein level rather than the mRNA level. Collectively, these results suggested that as a tumor suppressor, miR-24-1 may regulate mouse hepatocarcinoma cells migration and invasion, at least partially through targeting OGT, which could regulate the O-GlcNAcylation and the stability of this oncoprotein c-Myc. This may give insight into a novel mechanism and therapy of tumor lymphatic metastasis.