Pancreatic adenocarcinomas are aggressive and frequently develop resistance to all current therapies. Replication-selective adenoviruses can overcome resistance to chemotherapeutics through their sensitizing effects on drug-induced cell killing. We previously found that adenovirus deleted in the anti-apoptotic E1B19K gene enhanced gemcitabine-induced apoptotis. Here we demonstrate that our engineered double-deleted AdDD mutant (deleted in the pRb-binding E1ACR2 region and E1B19K) selectively replicates and enhances cell killing in combination with DNA-damaging cytotoxic drugs in pancreatic cancer cells. Combinations of AdDD with gemcitabine, irinotecan or cisplatin resulted in two-to fourfold decreases in EC 50 (half maximal effective concentration) values and was more efficent than similar combinations with wild-type virus, the dl1520 (ONYX-015) and dl922-947 mutants. AdDD replication was impaired in normal bronchial human epithelial cells and did not sensitize the cells to drugs. Gemcitabine-insensitive AsPC-1, BxPC-3 and PANC-1 cells were efficiently killed by irinotecan in combination with AdDD. Suboptimal doses of AdDD and gemcitabine significantly prolonged time to tumor progression in two human pancreatic tumor xenograft in vivo models, PT45 and SUIT-2. We conclude that AdDD has low toxicity to normal cells while potently sensitizing pancreatic cancer cells to DNA-damaging drugs, and holds promise as an improved therapeutic strategy for pancreatic cancer.