Gemcitabine is first-line therapy for advanced pancreatic ductal adenocarcinoma (PDAC) with a poor survival and response rate. Hyperbaric oxygenation (HBO) enhances delivery of oxygen to hypoxic tumor cells and increases their susceptibility to cytotoxic effects of chemotherapy. We hypothesized that the anticancer activity of gemcitabine (GEM) may be enhanced if tumor cells are placed in an oxygen-rich environment. The present study evaluated the effects of gemcitabine, HBO and their combination on apoptosis of tumor cells. Materials and Methods: PANC-1 and AsPc-1 PDAC tumor cell lines wereused. Cultured tumor cells were treated with GEM at its growth-inhibitory concentration (IC50) and HBO at 2.5 ATA for 90 min or a combination of both (HBO then GEM and GEM then HBO). Twenty-four hours later, apoptotic cells ineach group were analyzed and the apoptotic index (AI) wascalculated. Results: PANC-1 cell line: HBO alone had noeffect on AI: 6.5\ub10.1 vs. 5.9\ub10.1. HBO before and aftergemcitabine did not further increase AI: 8.2\ub10.1 (HBOGEM),8.5\ub10.1 (GEM-HBO) vs. 8.1\ub10.1 (GEM). The combination of HBO and gemcitabine significantly increased AI: 10.7\ub10.02 (p<0.001 vs. all groups). AsPc-1 cell line: HBO-alone had no effect on AI: 5.9\ub10.1 vs. 5.9\ub10.1. HBO before and after gemcitabine did not further increase AI:8.2\ub10.1 (HBO-GEM), 8.4\ub10.1 (GEM-HBO) vs. 8.0\ub10.1 (GEM). The combination of HBO and gemcitabine significantly increased AI: 9.7\ub10.1 (p<0.001 vs. all groups). Conclusion: HBO-alone, whether administered before and after gemcitabine has no effect on apoptosis of PDAC cellsin vitro. HBO significantly enhanced gemcitabine-induced apoptosis when administered during gemcitabine. Our findings suggest that the time window would be critical for using HBO as adjuvant to chemotherapy