Multimodal approaches combining drugs that differentially function is the most popular regimen for treating human cancer. Understanding the molecular mechanisms underlying the synergistic, potentiative, and antagonistic effects of drug combinations could facilitate the discovery of novel efficacious combinations. We previously showed that telomerase-specific replication-competent adenovirus (Telomelysin, OBP-301), in which the human telomerase reverse transcriptase promoter controls the adenoviral E1 gene expression, induces a selective antitumor effect in human cancer cells. Here, using E1-deleted replication-deficient adenovirus expressing the p53 tumor suppressor gene (Advexin, Ad-p53) and OBP-301, we investigate how these adenoviruses that kill tumor cells with different mechanisms could work in combination on human cancer. We found that E1-deficient Ad-p53 could kill cancer cells more efficiently in the presence of OBP-301 than Ad-p53 alone or OBP-301 alone, because Ad-p53 could become replication-competent by being supplied adenoviral E1 from coinfected OBP-301 in trans. Ad-p53 plus OBP-301 induced high levels of p53 protein expression without p21 induction, resulting in apoptotic cell death documented by active caspase-3 expression with a cytometric bead array and an increased subdiploid apoptotic fraction of the cell cycle. For in vivo evaluation, nude mice xenografted with human lung tumors received intratumoral injection of OBP-301 and/or Ad-p53. Analysis of the growth of implanted tumors showed an enhanced antitumor effect in combination therapy. Our data show that Ad-p53 in combination with OBP-301 induces not only oncolytic but also apoptotic cancer cell death and enhances antitumor activity in vitro and in vivo, providing potential merits as a multimodal treatment for human cancer.