Calcium channel blockers including verapamil have been proposed to enhance release and antitumor efficacy of oncolytic adenoviruses in preclinical studies but this has not been studied in humans before. Here, we studied if verapamil leads to increased replication of oncolytic adenovirus in cancer patients, as measured by release of virions from tumor cells into the systemic circulation. The study was conducted as a matched case-control study of advanced cancer patients treated with oncolytic adenoviruses with or without verapamil. We observed that verapamil increased mean virus titers present in blood after treatment (P < 0.05). The frequency or severity of adverse events was not increased, nor were cytokine responses or neutralizing antibody levels different between groups. Signs of possible treatment-related clinical benefits were observed in both groups, but there was no significant difference in responses or survival. Thus, our data suggests that the combination of verapamil with oncolytic adenoviruses is safe and well tolerated. Moreover, verapamil treatment seems to result in higher virus titers in blood, indicating enhanced overall replication in tumors. A randomized trial is needed to confirm these findings and to study if enhanced replication results in benefits to patients.