Gene-attenuated replication-competent adenoviruses are emerging as a promising new modality for the treatment of cancer. For the aim of improving adenoviral vectors for cancer gene therapy, we have constructed genetically attenuated adenoviral vectors with different combinations of E1B genes and investigated the possibility of enhanced oncolytic and replication effects of these engineered replication-competent adenoviruses. We show here that the cytolytic potency of each gene-attenuated replicating adenovirus differed significantly depending on the presence or deletion of E1B 55 kDa and E1B 19 kDa function. More specifically, among the constructed vectors (Ad-deltaE1B19, Ad-deltaE1B55, Ad-deltaE1B19/55, and Ad-wt), E1B 19 kDa-inactivated adenovirus (Ad-deltaE1B19) was the most potent against all tumor cells tested, inducing the largest-sized plaques and marked CPE. Further, cells infected with either Ad-deltaE1B19 or E1B19/55 kDa-deleted adenovirus (Ad-deltaE1B19/55) showed complete cell lysis with disintegrated cellular structure, whereas cells infected with Ad-wt maintained intact cellular and nuclear membrane with properly structured organelles. TUNEL and DNA fragmentation assay also revealed that the Ad-deltaE1B19 or Ad-deltaE1B19/55 adenovirus-infected cells showed more profound induction of apoptosis in comparison to wild-type adenovirus-infected cells. The presence of E1B 55 kDa gene was required for efficient viral replication and deletion of E1B 19 kDa function in replicating adenovirus-induced apoptosis, leading to increased cytopathic effects. Moreover, Ad-deltaE1B19 adenovirus showed a better antitumor effect than other E1B-attenuated adenoviruses. Taken together, the replicating adenoviruses deleted in E1B 19 kDa function may serve as an improved vector for anticancer gene therapy in combination with apoptosis-inducing modalities such as chemotherapeutic agents and radiation therapy.