Resistance to gemcitabine is common and critically limits its therapeutic efficacy in patients with pancreatic cancer. Interferon‑beta (IFN‑β) induces numerous antitumor effects and synergizes with gemcitabine treatment. The immunomodulatory effects of this treatment regimen have not yet been described. In the present study, the antitumor effect of IFN‑β combined with gemcitabine was investigated in immune competent mice. Mouse KPC3 cells were used in all experiments. Treatment effects were determined with cell proliferation assay. Reverse transcription‑quantitative PCR was used to measure gene expression. For in vivo experiments, cells were subcutaneously injected in immune competent mice. For immune profiling, NanoString analysis was performed on tumor samples of treated and untreated mice. Baseline expression of Ifnar‑1 and Ifnar‑2c in KPC3 cells was 1.42±0.16 and 1.50±0.17, respectively. IC50 value of IFN‑β on cell growth was high (>1,000 IU/ml). IFN‑β pre‑treatment increased the in vitro response to gemcitabine (1.3‑fold decrease in EC50; P<0.001). In vivo, tumor size was not statistically significant smaller in mice treated with IFN‑β plus gemcitabine (707±92 mm3 vs. 1,239±338 mm3 in vehicle‑treated mice; P=0.16). IFN‑β alone upregulated expression of numerous immune‑related genes. This effect was less pronounced when combined with gemcitabine. For the first time, to the best of our knowledge, the immunomodulatory effects of IFN‑β, alone and combined with gemcitabine, in pancreatic cancer were reported. Prognostic markers for predicting effective responses to IFN‑β therapy are urgently needed.