Metastasis is the main cause of cancer-associated death, and metastasis of pancreatic cancer remains difficult to treat because of its aggressiveness. MicroRNAs (miRNAs) play crucial roles in the regulation of various human transcripts, and many miRNAs have been reported to correlate with cancer metastasis. We identified an anti-metastatic miRNA, miR-5100, by investigating differences in miRNA profiling between highly metastatic pancreatic cancer cells and their parental cells. Overexpression of miR-5100 inhibited colony formation (P<0.05), cell migration (P<0.0001) and invasion (P<0.0001) of pancreatic cancer cells. In addition, we identified a possible target of miR-5100, podocalyxin-like 1 (PODXL), and demonstrated miR-5100 directly binds to the 3' untranslated region of PODXL and post-transcriptionally regulates its expression in pancreatic cancer cells. Silencing PODXL resulted in diminished cell migration (P<0.0001) and invasion (P<0.05). We also clarified the close relationship between expression of PODXL in human pancreatic cancer specimens and liver metastasis (P=0.0003), and determined that post-operative survival was longer in the low-PODXL expression group than in the high-PODXL expression group (P<0.05). These results indicate that miR-5100 and PODXL have considerable therapeutic potential for anti-metastatic therapy and could be potential indicators for cancer metastases in patients with pancreatic cancer.