Abstract: Objective: To improve the antineoplastic activity of suicide gene therapy and to investigated the antitumor mechanism of this therapy in breast cancer controlled by Tet regulation system. Methods: Recombinant retroviruses containing HSVtk and Tet-On gene were constructed and transduced into MCF-7 cells. Target gene was inserted into the host cells and performed by Southern-blotting. The antiproliferative effect on infected MCF-7 cells was detected by trypan-blue exclusion assay, observed morphological aspect of apoptosis by inverted fluorescent microscope and assessed apoptosis rate by flowcytometric method. To examine the therapy effect on the BALB/C nude mice of breast cancer under the Tet-On induction, retroviruses were injected into the tumors and pathological analysis was performed. Results: It was showed ganciclovir (GCV) can enhance tumor-killing activity in retrovirus-infected MCF-7 cells under higher induction of Doxycycline (DOX). After treatment of different concentration of DOX, inhibition rate of proliferation and apoptosis rate of MCF-7 cells were increased. Seven days after DOX induction and 30 days following GCV treatment, tumorous size of nude mice was decreased remarkably and apoptosis could be detected in tumor cells. Conclusions: Recombinant retrovirus containing Tet regulation system and HSVtk gene can successfully infect MCF-7 cells and induce apoptosis in vivo and in vitro of tumor cells.