Abstract Purpose: The purpose of this study was to assess the impact of anti-adenovirus neutralizing antibodies (AdNAbs) on the distribution, tolerability, and efficacy of intravenously administered oncolytic adenovirus. A translational model was developed to evaluate the impact of humoral immunity on intravenous administration of oncolytic adenovirus in humans. Experimental Design: Initially, severe combined immunodeficient (SCID)/beige mice were passively immunized with various amounts of human sera to establish a condition of preexisting humoral immunity similar to humans. A replication-deficient adenovirus encoding β-galactosidase (rAd-βgal) was injected intravenously into these mice. An AdNAb titer that mitigated galactosidase transgene expression was determined. A xenograft tumor-bearing nude mouse model was developed to assess how a similar in vivo titer would impact the activity of 01/PEME, an oncolytic adenovirus, after intravenous administration. Results: In SCID/beige mice, there was a dose dependence between AdNAbs and galactosidase transgene expression; 90% of transgene expression was inhibited when the titer was 80. A similar titer reconstituted in the nude mice with human serum, as was done in the SCID/beige mice, did not abrogate the antitumor efficacy of the replicating adenovirus after intravenous administration. Viral DNA increased in tumors over time. Conclusions: In intravenous administration, preexisting AdNAb titer of 80 significantly attenuated the activity of a 2.5 × 1012 particles per kilogram dose of nonreplicating adenovirus; the same titer had no affect on the activity of an equivalent dose of replicating adenovirus. Our results suggest that a majority of patients with preexisting adenovirus immunity would be candidates for intravenous administration of oncolytic adenovirus.