Although microRNA-21 (miR-21) is emerging as an oncogene and has been shown to target several tumor suppressor genes, including programmed cell death 4 (PDCD4), its precise mechanism of action on cancer stem cells (CSCs) is unclear. Herein, we report that FOLFOX-resistant HCT-116 and HT-29 cells that are enriched in CSCs show a 3- to 7-fold upregulation of pre- and mature miR-21 and downregulation of PDCD4. Likewise, overexpression of miR-21 in HCT-116 cells, achieved through stable transfection, led to the downregulation of PDCD4 and transforming growth factor beta receptor 2 (TGFβR2). In contrast, the levels of β-catenin, TCF/LEF activity and the expression of c-Myc, Cyclin-D, which are increased in CSCs, are also augmented in miR-21 overexpressing colon cancer cells, accompanied by an increased sphere forming ability in vitro and tumor formation in SCID mice. Downregulation of TGFβR2 could be attributed to decreased expression of the receptor as evidenced by reduction in the activity of the luciferase gene construct comprising TGFβR2-3' untranslated region (UTR) sequence that binds to miR-21. Moreover, we observed that downregulation of miR-21 enhances luciferase-TGFβR2-3' UTR activity suggesting TGFβR2 as being one of the direct targets of miR-21. Further support is provided by the observation that transfection of TGFβR2 in HCT-116 cells attenuates TCF/LEF luciferase activity, accompanied by decreased expression of β-catenin, c-Myc and Cyclin-D1. Our current data suggest that miR-21 plays an important role in regulating stemness by modulating TGFβR2 signaling in colon cancer cells.