Objective: To investigate the therapeutic effect and metabolism of ganciclovir in oral squamous carcinoma cells after being treated with adenovirus-mediated herpes simplex virus tymidine kinase (AdCMVHSV-TK)/ganciclovir (GCV) system. Methods: Human oral squamous carcinoma cells (Tca8113 cell line) and xenografts in BALB/c nude mice were treated with the AdCMVHSV-TK/GCV system. The killing effect in vitro by MTT assay was detected and the tumor inhibition effect observed. High-performance liquid chromatography (HPLC) was performed to determine the metabolism of GCV in vitro and in vivo. Results: The AdCMVHSV-TK/GCV system had a strong killing effect on Tca8113 cells. In cell extracts there were two peaks identified as GCV and phosphorylated GCV by HPLC. Compared with the control groups, mice treated with the AdCMVHSV-TK/GCV system demonstrated significant tumor regression (P < 0.001); the tumor was doubling time prolonged and the inhibition rate was 90.69%. There were GCV and phosphorylated GCV in tumor tissues, but only GCV was found in blood. Conclusions: The AdCMVHSF-TK/GCV suicide gene system had a significant in vitro and in vivo antitumor effect on oral squamous cell carcinoma due to converting GCV into phosphorylated GCV.