Background/Aim: Gemcitabine (GEM) is used in clinical chemo-radiotherapy; however, the mechanism that contributes to enhanced radiosensitivity by GEM is not fully-understood. We evaluated the effect of GEM on radiosensitization in pancreatic cancer cell lines. Materials and Methods: Pancreatic cell lines PK-59 and PK-45p were used. A total of 5 mu M GEM for 4 h were administered pre- or post-gamma irradiation. Results: Enhanced cell killing effects by GEM in radiotherapy were observed for pre-treatment but not post-treatment GEM. We focused on the dynamics of RAD51 and phospho-H2AX foci after irradiation. Significantly higher numbers of phospho-H2AX foci were observed in GEM pre-treated cells than in untreated cells after irradiation. We also found inhibition of the formation and degradation of RAD51 foci by GEM pre-treatment. The radiosensitizing effect of GEM was suppressed by knockdown of RAD51. Conclusion: RAD51-dependent homologous recombination is one of the key targets in the GEM-induced radiosensitizing effect.