Pancreatic ductal adenocarcinoma (PDAC) is a devastating disease with a cumulative 5-year overall survival of less than 5% for all stages. Thirty percent of patients diagnosed with PDAC present with a locally advanced disease could benefit from chemoradiotherapy with gemcitabine, which is effective but toxic. Over the past few years, studies have focused on the development of targeted radiosensitizer such as poly(ADP-ribose) polymerase (PARP) inhibitor. We conducted an in vitro study to determine whether PARP inhibition enhances radiation-induced cytotoxicity of PDAC cell lines. Three human pancreatic cancer lines, MIA PaCa-2, AsPC-1 and PANC-1 were treated in their exponential growth phase at room temperature. Low-LET gamma radiations were carried out using a 137Cs irradiator (Biobeam GM 8000) with a dose rate of 3.4 Gy/min and increasing doses of 2, 5 and 10 Gy. Cells were previously treated with non-cytotoxic concentration of gemcitabine (10 nM) and/or olaparib (1 μM). Clonogenic assay, cell cycle distribution and γ-H2AX quantifications were performed. In vitro , treatment with olaparib alone was not cytotoxic but highly radiosensitized cell lines (standard enhancement ratio (SER) = 1.21±0.06 for AsPc-1, SER = 1.27±0.02 for MIA PaCa-2, and SER=1.38±0.01 for PANC-1), particularly at high dose per fraction (10 Gy). In contrast, a non-cytotoxic concentration of gemcitabine also radiosensitized cells, but clearly less than olaparib (SER= 1.22±0.01 for AsPc-1, SER=1.11±0.04 for MIA PaCa-2 and SER=1.03±0.04 for PANC-1). Interestingly, gemcitabine and olaparib combination had a synergistic radiosensitive effect on PANC-1 cells, but not on MIA PaCa-2 cells. AsPC-1 cell line was radiosensitive with high irradiation dose, and no colonies were formed after 10 Gy. In addition, combination of olaparib and irradiation induced a G2/M arrest in all tested cell lines. Radiosensitization by gemcitabine was associated with percentage of cells blocked in early S-phase. Finally, 24h after treatment, the number of remaining γ-H2AX stained cells was higher with cells treated with a combination of olaparib and 10 Gy irradiation, compared to irradiation or olaparib alone. Our results showed that MIA PaCa-2, AsPC-1 and PANC-1 cell lines could be radiosensitized by the combination of olaparib, through an increase of unrepaired double-strand breaks and a block in G2 phase. The radiosensitization was increased with high dose of radiation. This combination of olaparib with gemcitabine based chemoradiotherapy could lead to an enhancement of local control in vivo and an improvement in disease-free survival.