Oncolytic viruses are an excellent platform for developing effective strategies in cancer immunotherapy. Several challenges remain in the use of viro-immunotherapy for cancer, such as the lack of costimulatory signals and negative regulation of immune checkpoints. In this study, we designed a novel adenovirus expressing a soluble fusion protein, PD-1/CD137L, which contains the extracellular domains of PD-1 and CD137L at each terminus (Ad5-PC). Ad5-PC preserved the costimulatory activity of CD137L and facilitated the persistence of activated CD8+ T cells. Ad5-PC induced strikingly increased antitumor activity in both ascitic and subcutaneous HCC tumor models, with 70% and 60% long-term cure rates, respectively. The improved antitumor effect of Ad5-PC was attributed to the sustained high-level lymphocyte activation and IFN-γ production in the tumor microenvironment and was essentially dependent on CD8+ T cells rather than NK cells. Moreover, Ad5-huPC expressing human soluble PD-1/CD137L fusion protein was effective in suppressing tumor growth and improving survival in a humanized mouse model. We confirmed that Ad5-PC induced tumor-specific and systematic protection against tumor rechallenges at both in situ and at distant sites. Thus, Ad5-PC harnesses several distinct functions to efficiently overcome several major hurdles of viro-immunotherapy.