Due to invasion and intrahepatic metastasis, the prognosis for patients with hepatocellular carcinoma (HCC) is poor. However, the mechanisms underlying these processes of HCC remain unclear. Cancer stem cells may be involved in early systemic dissemination and metastasis formation and side population (SP) cells isolated from diverse cancer cells possess stem cell-like properties. However, the mechanisms involved in migration and invasion of cancer stem cells are not well understood. In this study, we identified and isolated populations of SP cells from HCC cell lines using flow cyto-metry. SP cells showed higher levels of migration and invasion capability. Higher expression of miR-21 was observed in SP cells. Silencing of miR-21 led to a reduction in the migration and invasion of these cells and overexpression of miR-21 can increase in cell migration and invasion. Overexpression of miR-21 did not cause degradation of PTEN or RECK or PDCD4 mRNA but drastically inhibited its protein expression. Consistent with these results, silencing miR-21 increased the levels of PTEN, RECK and PDCD4 protein, respectively. The role of silencing miR-21 was partially attenuated by silencing of PTEN or RECK or PDCD4 mRNA. The results of this study revealed the aberrant expression of miR-21 in SP cells and showed that miR-21 regulates the expression of multiple target proteins that are associated with tumor dissemination. MiR-21 is a pro-metastatic miRNA in SP cells and raises the possibility that therapy of HCC may be improved by pharmaceutical strategies directed towards miR-21.