Background: MicroRNAs play an essential role in colorectal cancer development and progression. Aberrant miR-409-3p expression has been reported in several cancers. However, the clinical significance and functions of miR-409-3p in human CRC were not entirely clear. Methods: miR-409-3p expression levels were determined in 45 pairs of primary CRC and their corresponding adjacent non-tumor tissues by qPCR. The effects of ectopic expression of miR-409-3p on CRC cells proliferation, wound healing, metastasis were investigated by CCK-8, transwell assay and peritoneal spreading nude mice model. Results: Statistical analysis of clinical cases revealed that low miR-409-3p expression had inclinations towards lager tumor size and local invasion. Ectopic expression of miRNA mimics suggested that miR-409-3p could inhibits the abilities of proliferation, wound healing, metastasis and invasion in CRC cells. Notably, we found the NLK could be a potential target of miR-409-3p. Conclusion: Our results suggest that miR-409-3p functions as a tumor suppressor by inhibiting the development and metastasis of CRC, suggesting that miR-409-3p is expected to become a new diagnostic marker and a new target of the treatment of CRC.