microRNAs are implicated in cancer initiation and progression by their ability to affect the expression of genes and proteins that regulate cell proliferation and death. Recent studies found that the stem cell-related genes Sox2, Oct4 and Klf4 are among the target genes regulated by microRNA-145 (miR-145), suggesting that miR-145 possibly plays a role in the maintenance of cancer stem cells. Therefore, it is important to address the involvement of miR-145 in the key roles of cancer stem cells in cancer initiation, progression and reoccurrence. We compared miR-145 expression in the cancer stem-like cells (T3A-A3) derived from hepatocarcinoma, in the hepatocarcinoma cell line BEL-7402 and in the normal liver sinusoidal endothelial cell line (LSEC). As demonstrated by a TaqMan microRNA real-time assay, T3A-A3 cells express lower miR-145 levels compared to the other cell lines. To address the role of miR-145 in cancer stem cells, miR-145 was restored in T3A-A3 cells. This resulted in senescence-like G, arrest in cell cycling, and significantly inhibited clonogenic cell expansion in vitro and xenograft tumor growth in vivo. Moreover, miR-145 restoration diminished tumorsphere growth of T3A-A3 cells in vitro and T3A-A3 cells tumor formation in nude mice in vivo. Additionally, the increase in miR-145 levels paralleled the decrease in Oct4 levels. The effect of miR-145 on tumor suppression in T3A-A3 cells was partly reversed by overexpression of Oct4 both in vitro and in vivo. Collectively, our data indicate that miR-145 plays an important role in cancer stem cell tumorigenicity, potentially via modulation of the downstream target, Oct4.