Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal malignancies, and chemotherapy is still an important treatment. It is urgent to develop new medicines because of the limitation and side effects of chemotherapy. 2′-Hydroxyflavanone (2HF) is a citrus-bioflavonoid that is considered to have anti-cancer efficacy. Compared to human pancreatic ductal epithelial cells hTERT-HPNE, more significant growth-inhibitory effects were seen in PDAC cells BxPC-3 and MIA PaCa-2. We showed that apoptosis was induced and that the cell cycle was arrested when cells were treated with 2HF. The expression of the molecular proteins cleaved PARP, cleaved Caspase3, Bax, Bcl-2, CyclinD1, and p27 changed correspondingly. Also, we observed anti-metastatic effects and changes in MMP9, E-cadherin, N-cadherin and Vimentin when cells were treated with a low dose of 2HF. Suppression of STAT3 and EGFR phosphorylation was also identified as a result of treatment with a combination of 2HF and EGFR inhibitors. The in vivo antitumor effects in KPC mice were consistent with those observed in vitro. 2HF has impactful anti-cancer efficacy and sensitizes human pancreatic cancer cells to EGFR inhibitors through the inhibition of STAT3. 2′-Hydroxyflavanone (2HF), a natural flavonoid extract from oranges and citrus fruits, was confirmed to have antitumor effects in pancreatic cancer in vivo and in vitro.•2HF inhibited the viability and clonogenicity of pancreatic cancer cells•2HF induced apoptosis of pancreatic cancer cells•2HF caused the cell cycle arrest of pancreatic cancer cells•2HF suppressed the invasion and migration of pancreatic cancer cells•2HF enhanced pancreatic cancer cell chemosensitivity by inhibiting activation of STAT3