Octamer-binding transcription factor 4 (OCT4) was closely related to pancreatic cancer progression, but its regulation in pancreatic cancer by microRNA (miRNA) is not fully clear. OCT4-positive and OCT4-negative pancreatic cells were isolated by flow cytometry, and it was found that OCT4-positive cells are enriched in transplanted pancreatic cancer cells compared with the primary ones and showed increasing proliferation and sphere formation. The data of miRNA array assay showed that miR-335 in OCT4-positive pancreatic cancer cells was lower than that in the negative ones. The results were confirmed in pancreatic cancer tissue and cell lines. Through expression analysis, it was found that miR-335 was underexpressed in OCT4(+) pancreatic cancer cells purified from primary tumors. Enforced expression of miR-335 in OCT4(+) pancreatic cancer cells inhibited clonogenic expansion and tumor development. miR-335 re-expression in OCT4(+) pancreatic cancer cells was blocked. Systemically delivered miR-335 inhibited pancreatic cancer metastasis and extended animal survival. Of significance, OCT4 was identified and validated as a direct and functional target of miR-335. Taken together, our results provide evidence that miR-335 might inhibit progression and stem cell properties of pancreatic cancer targeting OCT4.