Cervical cancer is one of the most common malignant tumors among women worldwide. In some regions, its incidence and mortality rates remain high, and patients with HPV-unassociated tumors often have a poor prognosis. Those with advanced or recurrent disease frequently show unsatisfactory treatment outcomes due to metastasis and chemoresistance. Ribosome biogenesis is notably active in various cancer cells and has emerged as a potential therapeutic target. CX-5461, a specific inhibitor of RNA polymerase I, was investigated in this study for its therapeutic effect and underlying mechanism in cervical cancer. The results demonstrated that CX-5461 significantly inhibits the proliferation of cervical cancer cells, activates the ATM/ATR pathway, and induces DNA damage. Furthermore, it leads to abnormal accumulation of Cyclin B1 and aberrant activation of phospho-CDK1-T161, driving cells with DNA damage into mitosis. This process ultimately triggers mitotic catastrophe, resulting in cell death or senescence. When combined with cisplatin, CX-5461 enhances the sensitivity of cervical cancer cells to this chemotherapeutic agent. In conclusion, CX-5461 demonstrates potential therapeutic value for cervical cancer, particularly as a new strategy for patients with primary or platinum-resistant disease.