Cancer stem cells (CSCs) have the characteristics of self-renewal, unlimited proliferation, and initiating new tumors. However, the origin of CSCs is still controversial. F6, a tumor cell line transformed from human fetal mesenchymal stem cells, was established in our previous study. Whether CSCs exist in this mutated cell line remains unclear. In the present study, we isolated CSCs from F6 cells based on CD133 expression using flow cytometry and investigated the biological characteristics of CD133+ F6 cells (F6-CD133+). We observed that the F6-CD133+ cells grew faster and had a higher capacity of colony formation than the F6-CD133- cells and parental F6 cells in vitro. In addition, F6-CD133+ cells had a higher tumorigenic ability than F6-CD133- cells in vivo since 1,000 F6-CD133+ cells were able to form tumors in nude mice. Cell viability assay revealed that F6-CD133+ cells were more sensitive to cisplatin while less to 5-fluorouracil. Furthermore, gene expression profile analysis showed that there were 673 differentially expressed genes between F6-CD133+ and F6-CD133- cells, many of which were involved in key cell signaling pathways. Taken together, our findings confirm that F6 cells contain a population of CSCs that contribute to its heterogeneity and tumorigenic potential, indicating that transformed stem cells could be the source of CSCs, and targeting this population may lead to more effective tumor treatments.