The present study aimed to investigate the effects of obatoclax (OBX) combined with gemcitabine (GEM) treatment on the proliferation, migration, invasion and epithelial‑mesenchymal transition (EMT) related proteins of pancreatic cancer cell line BxPC‑3 under hypoxic conditions. Protein expression levels of hypoxia‑inducible factor 1α (HIF‑1α) in BxPC‑3 pancreatic cancer cells under normoxic and hypoxic conditions were detected by western blotting. Cells were divided into four groups: Normoxia group, hypoxia group, OBX group and OBX + GEM group. The proliferation activity of BxPC‑3 cells was detected by Cell Counting kit‑8. The migratory and invasive abilities of BxPC‑3 cells were detected by the scratch test and Matrigel assay, respectively. The protein expression levels of vimentin, E‑cadherin and p53 in BxPC‑3 cells were also detected by western blotting. HIF‑1α expression under hypoxic conditions was significantly increased compared with expression under normoxic conditions. Under hypoxic conditions, OBX treatment reduced cell activity, decreased cell migration and invasion, promoted the expression of E‑cadherin and p53. In the OBX + GEM group, BxPC‑3 cell activity decreased significantly, cell migration and invasion decreased significantly, the expression of vimentin was significantly reduced and the expression of E‑cadherin and p53 further increased. In conclusion, the present results demonstrated that under hypoxic conditions, OBX combined with a small dose of GEM may be able to inhibit the growth, migration and invasion of pancreatic cancer cells, possibly via inhibition of EMT process. These results may provide a promising strategy for pancreatic cancer therapy.