MicroRNAs (miRNAs) regulate tumor progression and invasion via direct interaction with target messenger RNAs (mRNAs). We defined miRNAs involved in cancer metastasis (metastamirs) using an established in vitro colorectal cancer (CRC) model of minimally metastatic cells (SW480 line) from a colon adenocarcinoma primary lesion and highly metastatic cells (SW620 line) from a metastatic lymph node from the same patient 1 year later. We used microarray analysis to identify miRNAs differentially expressed in SW480 and SW620 cells, focusing on miR-499-5p as a novel candidate prometastatic miRNA whose functions in cancer had not been studied. We confirmed increased miR-499-5p levels in highly invasive CRC cell lines and lymph node-positive CRC specimens. Furthermore, enhancing the expression of miR-499-5p promoted CRC cell migration and invasion in vitro and lung and liver metastasis in vivo, while silencing its expression resulted in reduced migration and invasion. Additionally, we identified FOXO4 and PDCD4 as direct and functional targets of miR-499-5p. Collectively, these findings suggested that miR-499-5p promoted metastasis of CRC cells and may be useful as a new potential therapeutic target for CRC.