A variety of oncolytic viruses (OVs) are being tested in clinical trials for different human cancers. Although the innate immune response is critical as the first line of defense in thwarting viral infection of mammalian cells, little is known of this response in the context of OV therapy of tumors. Investigations of activities against a herpes simplex OV demonstrated that HSV-seronegative sera from rats, mice, and humans efficiently neutralize this OV in vitro. Although this neutralization is due to complement, activation of this innate host defense differs in its pathways among species routinely used in preclinical tumor trials. In rats, both natural immunoglobulins and mannan-binding lectin (MBL) activate complement against the OV, while in mice only MBL is relevant to this activation. However, in humans only natural immunoglobulins play a role in complement activity. Quantitative analyses confirm that in vivo complement depletion facilitates the initial infection of tumors by systemic OVs. Therefore, complement activation against oncolytic HSV vectors proceeds through different pathways in different species. These findings are relevant to preclinical rodent studies of OV therapy and their application to human clinical trials.