Shikonin is a traditional Oriental medical herb extracted from Lithospermum erythrorhizon. Many studies have shown that shikonin possesses anticancer ability against many different cancers, including hepatocellular carcinoma (HCC). Recently, tumor metastasis has been become an important clinical obstacle. However, the effect of shikonin on metastasis by HCC is unknown. The 50% inhibitory concentration (IC50) of shikonin on HCC cells was determined by an MTT assay and the xCELLigence biosensor system. The migratory ability of HCC cells was detected by a transwell migration assay and the xCELLigence biosensor system. Matrix metalloproteinase-2 and -9 (MMP-2 and -9) expression levels were determined by Western blotting, and the activities of MMP-2 and -9 were determined by gelatin zymography. We found that IC50 values of HepJ5 and Mahlavu cells to shikonin treatment were around 2 μM. Exposure to a low dose of shikonin (0-0.4 μM) did not influence the survival of HCC cells. Interestingly, exposure to a low dose of shikonin inhibited the migratory ability on HepJ5 and Mahlavu cells. To further dissect the mechanism, we found that treatment with a low dose of shikonin reduced the activities and expression levels of MMP-2 and -9, which were correlated with the decreased cell migratory ability of HCC cells. In addition, we found a decrease of vimnetin expression, but no influence on the expression levels of N-cadherin, TWIST, or GRP78. In mechanism dissecting, we found that shikonin treatment may suppress the phosphorylation of AKT and then reduce the NF-κB (NF = nuclear factor) levels, but has no influence on the levels of c-Fos and c-Jun. Furthermore, we also found that shikonin may also reduce the phosphorylation of IκB. We concluded that a low dose of shikonin can suppress the migratory ability of HCC cells through downregulation of expression levels of vimentin and MMP-2 and -9. Our findings suggest that shikonin may be a new compound to prevent the migration of HCC cells.