Tumor-selective replicating viruses are attractive tools for cancer gene therapy, but generally achieve only transitory tumor suppression. However, replicating retrovirus vectors (RRVs) can achieve highly efficient and tumor-selective transduction, as well as persistent expression of transgenes. We therefore developed RRVs that express the yeast cytosine deaminase (yCD) and herpes simplex virus thymidine kinase (TK), which exhibit remarkably enhanced cytotoxicity after administration of the prodrugs 5-fluorocytosine (5-FC) and ganciclovir (GCV) concomitant with the efficiency of their replicative spread, and tested their therapeutic effect in vitro and in vivo. In subcutaneous MDA-MB-435 human breast cancer xenograft models, RRV-mediated yCD and TK suicide gene therapy significantly suppressed tumor growth after prodrug administration. Notably, no systemic spread of the vector to extratumoral tissues was detected. Our results thus demonstrate that efficient, tumor-selective, and stable integration achieved by RRVs causes efficient cell killing upon prodrug administration, resulting in significant suppression of tumor growth.