miR-543 has been implicated as having a critical role in the development of breast cancer, endometrial cancer and hepatocellular carcinoma. However, the exact clinical significance and biological functions of miR-543 in colorectal cancer (CRC) remain unclear. Here, we found that miR-543 expression significantly downregulated in tumors from patients with CRC, APC Min mice and a mouse model of colitis-associated colon cancer. miR-543 level was inversely correlated with the metastatic status of patients with CRC and the metastatic potential of CRC cell lines. Moreover, ectopic expression of miR-543 inhibited the proliferation and metastasis of CRC cells in vitro and in vivo by targeting KRAS, MTA1 and HMGA2. Conversely, miR-543 knockdown promoted the proliferation, migration and invasion of CRC cells in vitro and augmented tumor growth and metastasis in vivo. Furthermore, we found that miR-543 expression was negatively correlated with the levels of KRAS, MTA1 and HMGA2 in clinical samples. Collectively, these data show that miR-543 inhibits the proliferation and metastasis of CRC cells by targeting KRAS, MTA1 and HMGA2. Our study highlights a pivotal role for miR-543 as a suppressor in the regulation of CRC growth and metastasis and suggests that miR-543 may serve as a novel diagnostic and prognostic biomarker for CRC metastasis.