ω3-polyunsaturated fatty acids (ω3- PUFAs) are known to possess anticancer properties. However, the relationship between ω3-PUFAs and β-catenin, one of the key components of the Wnt signaling pathway, in human pancreatic cancer remains poorly characterized. Human pancreatic cancer cells (SW1990 and PANC-1) were exposed to two ω3-PUFAs, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), to investigate the relationship between ω3-PUFAs and the Wnt/β-catenin signaling pathway in vitro. Mouse pancreatic cancer (PANC02) cells were implanted into fat-1 transgenic mice, which express ω3 desaturases and result in elevated levels of ω3-PUFAs endogenously. The tumor size, levels of Wnt/β-catenin signaling molecules and apoptosis levels were analyzed to examine the influence of ω3-PUFAs in vivo. DHA and EPA significantly inhibited cell growth and increased cell death in pancreatic cancer cells. DHA also reduced β-catenin expression, T cell factor/lymphoid-enhancing factor reporter activity and induced β-catenin/Axin/GSK-3β complex formation, a known precursor to β-catenin degradation. Furthermore, Wnt3a, a natural canonical Wnt pathway ligand, reversed DHA-induced growth inhibition in PANC-1 cells. Immunohistochemical analysis showed aberrant upregulation and increased nuclear staining of β-catenin in tumor tissues from pancreatic cancer patients. However, β-catenin levels in tumor tissues from fat-1 transgenic mice were reduced with a significant increase in apoptosis compared with those from control mice. ω3-PUFAs may be an effective therapy for the chemoprevention and treatment of human pancreatic cancer. and IAP.