Adenovirus-mediated gene therapy shows remarkable promise as a new strategy for advanced pancreatic cancer, but satisfactory clinical results have not yet been obtained. To improve this gene therapy, we investigated the effects of gemcitabine (GEM) on transgene expression by adenoviral vectors and their biological effects. We used Ad-lacZ and adenoviral vector-expressing NK4 (Ad-NK4) as representative adenoviral vectors. These vectors express β-galactosidase (β-gal) and NK4 (which inhibits the invasion of cancer cells), respectively, under the control of the CMV promoter. Cells were infected with the individual adenoviruses and then treated with GEM. GEM increased β-gal mRNA expression and β-gal activity, and increased NK4 expression in both culture media and within infected cells, in dose-dependent manners. The increased expression of NK4 delivered by Ad-NK4 had biological effects by inhibiting the invasion of cancer cells. GEM also enhanced NK4 expression in SUIT-2 cells transfected with an NK4-expressing plasmid, suggesting that GEM enhanced CMV promoter activity. In in vivo experiments, NK4 expression within subcutaneously implanted tumors was increased in GEM-treated mice compared with control mice. These results suggest that adenovirus-mediated gene therapy with GEM may be a promising approach for treating pancreatic cancer, and that this combination therapy may decrease the risks of side effects.