Pancreatic adenocarcinoma has a poor prognosis and frequently develops resistance to standard chemotherapeutics. Oncolytic adenoviruses represent a promising approach to overcome treatment resistance. The replication-selective dl922-947 adenovirus, defective in pRb binding, targets cancers with deregulated cell cycle control, such as the majority of pancreatic tumors. Cell killing efficacy was higher for dl922-947 than for adenovirus type 5 (Ad5) and the clinically approved dl1520 in pancreatic cancer cells with K-ras, p16 and p53 mutations. Combinations of dl922-947 and 5-fluorouracil or gemcitabine (2'2'-difluoro-2-deoxytidine) resulted in strong synergistic cell killing in Suit-2 and the highly drug- and virus-resistant Hs766T cells. Viral uptake increased in response to drugs, but was independent of the expression levels of the viral attachment receptor coxsackie and adenovirus receptor (CAR), whereas expression levels of the internalization receptors α(v)β(3)- and α(v)β(5)-integrins were increased. Early viral E1A expression was potently induced with drugs contributing to the synergistic effects. The dl922-947 mutant was more efficacious than Ad5 in vivo in Hs766T and Suit-2 xenograft models. In combination with gemcitabine, median survival was further prolonged. We demonstrate that dl922-947 is highly efficacious in pancreatic cancers and conclude that oncolytic adenoviruses harboring the E1ACR2 deletion have great potential for development into future clinical candidates for pancreatic cancer.