The aim of the present study was to investigate the effects of PTEN and KAI1 gene overexpression on the proliferation, metastasis and radiosensitivity of ASPC‑1 pancreatic cancer cells under hypoxic conditions. Recombinant vectors that overexpress PTEN and KAI1 genes were transfected into hypoxic ASPC‑1 cells. Protein expression levels were detected by western blot analysis. An MTT cell growth curve assay and a colony‑forming assay were used to analyze cell proliferation, and a Transwell assay was performed to evaluate the metastatic ability of the cells. Annexin V flow cytometry was used to determinate the apoptotic rate of X-ray-treated ASPC-1 cells. Western blot analysis revealed that PTEN‑ and KAI1‑transfected ASPC-1 cells significantly upregulated PTEN and KAI1 expression. The proliferation of hypoxic ASPC-1 cells was significantly suppressed by PTEN and KAI1. Furthermore, PTEN and KAI1 overexpression inhibited the metastatic ability of hypoxic ASPC‑1 cells. Following X‑ray treatment, the percentage of apoptotic cells increased significantly in the ASPC-1 cells transfected with PTEN and KAI1, which demonstrated that hypoxic ASPC-1 cells were more radiosensitive due to PTEN and KAI1 overexpression. In conclusion, double overexpression of PTEN and KAI1 inhibited the proliferation and metastatic activity, and enhanced apoptosis induced by X‑ray in ASPC‑1 cells under hypoxic conditions, which indicates that PTEN and KAI1 double-expression may have valuable application in pancreatic cancer gene therapy.