Sprouty2 (Spry2) was identified recently as a tumor suppressor gene in cancer cells which inhibits the activation of receptor tyrosine kinases (RTKs). The present study explored the effect of Spry2 in colon cancer cells in order to assess its potential use in the treatment of colon cancer. Expression of Spry2 inhibited the growth of a colon cancer cell line, HCT116, and induced sensitization to fluorouracil (5-FU) and metformin. Spry2 promoted apoptosis of cancer cells in association with activation of the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) pathway and the blockade of Ras-Raf-Erk signaling. Treatment of Spry2-HCT116 cells with metformin resulted in a more prominent effect on the inhibition of cell migration. Inhibition of microRNA-21 (mir‑21) induced upregulation of Spry2 and PTEN which underscores the importance of mir-21 in Spry2-associated tumorigenesis of the colon. These results point toward a potential strategy for colon cancer treatment worthy of further investigation.