PURPOSE: Cervical cancer is the fourth most common cancer in women and the seventh most common of all human cancers. Development of new treatments is mandatory to improve the outcome of this disease. Replication-selective oncolytic herpes simplex viruses (HSVs) have emerged as a new platform for cancer therapy. The therapeutic potential of a triple-mutated oncolytic HSV (T-01) for human papillomavirus (HPV)-related cervical cancer was evaluated with immunodeficient and immune-complete models. METHODS: (1) The in vitro efficacy of T-01 on human cervical cancer cell lines, TC-1, HeLa, CaSki, and SKG IIIa was evaluated. (2) The in vivo efficacy of T-01 was examined in human HeLa xenograft and TC-1 syngeneic models of human cervical cancer. After flank tumors reached 5 mm in diameter, the first intratumoral (i.t.) administration of T-01 was performed. Intratumoral administration of T-01 was performed with a 5 day interval a total of 6 times. RESULTS: In the in vitro study, T-01 was highly cytotoxic for all cell lines (48 h after infection with T-01 at 1 × 105 PFU, T-01 killing HeLa: 67.5%, Caski: 62.8%, SKG IIIa: 43.2%). Furthermore, in the human HeLa xenograft and TC-1 syngeneic models, T-01 resulted in a significant reduction of tumor growth. In addition, tumor-bearing mice treated with T-01 showed significantly increased numbers of CD8 + T-cells precursors than the control mice (p = 0.03). CONCLUSIONS: These results demonstrate that T-01 has cytotoxic efficacy and inhibited against HPV-related cervical cancer cells. These findings indicate that T-01 has therapeutic potential for HPV-related cervical cancer.