Conditionally replication adenovirus M4, which was constructed in our lab, was proved to have good clinical application prospect for its good anti-tumor and anti-metastasis effect. However, clinically applying M4 faces many problems. One of the most important is the safety of M4. In this study, we investigated the safety of M4 by comparing with Adv-TK, which was proved to be safe in I-III phase clinical trials. M4 and Adv-TK were injected into mice via the tail vein separately, and the mice were sacrificed at the indicated time. Blood was collected for biochemical tests, the liver was harvested for hematoxylin and eosin (H&E) staining and viral quantification, and splenic lymphocytes were separated for adenovirus specific cellular immune response. Our results showed that M4 had no obvious effect on mouse general symptoms. A transient reversible infiltration of inflammatory cells in collect abbacy was only observed in M4 group, and a transient slight increase in Cr level was detected both after M4 and Adv-TK injection. The adenovirus specific cellular immune response induced by M4 was similar to that by Adv-TK, and the distribution and metabolism of M4 in the mouse liver were also similar to those of Adv-TK. It was concluded that conditionally replication adenovirus M4 had the same safety as Adv-TK. The study provides safety basis for the coming clinical trials of M4.