Oncolytic adenoviruses (Ad) represent a promising therapeutic platform for the treatment of cancer. Despite their reliable safety and ease of handling, the recognized oncolytic Ad immune responses, biodistribution, spreads and efficacy of viral replication, compared with non-replicating adenoviruses, in vivo, have not been well understood. The present study establishes a novel permissive mouse immunocompetent orthotopic colon carcinoma model focusing on non-replicating Adv-TKs, a well-established adjuvant treatment in cancer and comparing its antitumor efficacy and safety profile with those of the two newly synthesized oncolytic Ad vectors, M7 and M8, formed by the insertion of a fragment of HSV-TK cDNA into the deleted 6.7 K/gp19 K or ADP in the E3 region to the E1A CR2-deleted Ad5 vectors. Systemic administration in the permissive immunocompetent orthotopic colon carcinoma mouse model, which could better predict the clinical results of Ad-based treatment by combining the characteristics that fully mimic the clinical orthotopic carcinogenesis and permissive Ad replication, show M7 and M8, especially M8, to have more powerful antitumoral efficacy and similar safety profiles relative to the Adv-TK. This promises a significant advance in the field of Ad cancer therapy and will perhaps provide the impetus to design new preclinical M7 and M8 viruses in the future.