FOXO1, also known as FKHR, is a member of the Forkhead transcription factor family. Our previous study revealed that FOXO1 expression is significantly downregulated in pancreatic ductal adenocarcinoma (PDAC). However, our knowledge on the clinical significance of FOXO1 and its biological roles and associated mechanisms in PDAC tumorigenesis remains limited. In this study, we confirmed that FOXO1 is commonly downregulated in PDAC tissues, at both the mRNA and protein levels, compared to adjacent tissues. Furthermore, FOXO1 inhibited cell proliferation and tumor formation both in vitro and in vivo, and promoted pancreatic cancer cell invasion. Downregulation of FOXO1 resulted in enhanced Wnt/β-catenin signaling activity, thereby promoting cell proliferation and epithelial-mesenchymal transition. The highly expressed miR-27a could potentially be used to target the 3'-UTR of FOXO1 in PDAC tissues to inhibit or at least slow down the invasion and proliferation of cancerous cells. Taken together, our findings suggest that the miR-27a/FOXO1/β-catenin axis may serve as a promising therapeutic target in PDAC progression.