Colorectal cancer (CRC) is the most common cancer diagnosed worldwide, and the development of metastases is a major cause of mortality. Accumulating evidence suggests that microRNAs are important in carcinogenesis by affecting the expression of genes that regulate cancer progression. A number of studies have shown that miR-206 is frequently downregulated in many human malignancies, including CRC, and is associated with a more malignant phenotype. Previous studies involving HeLa and C2C12 cells have validated the inhibitory mechanism of miR-206 via NOTCH3 targeting. However, whether or not the interplay between miR-206 and NOTCH3 also occurs in CRC is unknown. Therefore, we investigated the tumor suppressive and metastatic effects of miR-206 and its target, NOTCH3, in CRC. Based on the inverse association between the expression of miR-206 and NOTCH3 in CRC tissues, miR-206 mimics were transiently transfected into the SW480 (and its metastatic strain) and SW620 colon cancer cell lines. Upregulation of miR-206 inhibited cancer cell prolife-ration and migration, blocked the cell cycle, and activated apoptosis. The tumor suppressive capacity of miR-206 had a similar effect on CRC cells, although with a different metastatic potential, and may be explained by direct NOTCH3 signaling inhibition and indirect cross-talk with other signaling pathways involving CDH2 and MMP-9. These results support miR-206 as a tumor suppressor in CRC and suggest a potential therapeutic target for clinical intervention.