We previously demonstrated that the downregulation of Casitas B-lineage lymphoma (c-Cbl) can sensitize tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in two different ways. One way is to block the rapid degradation of TRAIL receptors, which can sustain TRAIL-induced apoptosis for a long time. Here, we designed a replication-defective adenovirus expressing the short hairpin RNA (shRNA) against c-Cbl to test the possibility of developing a cancer gene therapy that can act as a sensitizer of TRAIL. As expected from the results of our previous study that used a stable cell line with downregulated c-Cbl, infection with the c-Cbl shRNA-expressing adenovirus led to an increase in the death receptor 4 (DR4) and DR5 levels, which is known to be a cause for the increase of TRAIL-induced apoptosis. In conclusion, we demonstrated that c-Cbl shRNA-expressing adenovirus is able to sensitize TRAIL-induced apoptosis in vivo as well as in vitro.