Metastatic osteosarcoma (OS) has a very poor prognosis. New treatments are therefore wanted. The conditionally replicative adenovirus Ad5-Delta24RGD has shown promising anti-tumor effects on local cancers, including OS. The purpose of this study was to determine whether intravenous administration of Ad5-Delta24RGD could suppress growth of human OS lung metastases. Mice bearing SaOs-lm7 OS lung metastases were treated with Ad5-Delta24RGD at weeks 1, 2 and 3 or weeks 5, 6 and 7 after tumor cell injection. Virus treatment at weeks 1-3 did not cause a statistically significant effect on lung weight and total body weight. However, the number of macroscopic lung tumor nodules was reduced from a median of >158 in PBS-treated control mice to 58 in Ad5-Delta24RGD-treated mice (p = 0.15). Moreover, mice treated at weeks 5-7 showed a significantly reduced lung weight (decrease of tumor mass, p < 0.05), a significantly increased body weight gain (decrease of disease symptoms, p < 0.005) and a reduced number of macroscopic lung tumor nodules (median 60 versus > 149, p = 0.12) compared to PBS treated control animals. Adenovirus hexon expression was detected in lung tumor nodules at sacrifice three weeks after the last intravenous adenovirus administration, suggesting ongoing viral infection. These findings suggest that systemic administration of Ad5-Delta24RGD might be a promising new treatment strategy for metastatic osteosarcoma.