Uterine serous carcinoma (USC) belongs to the non-endometrioid subtype of endometrial cancer that is known for its highly aggressive behavior and poor prognosis, highlighting the warrant of novel strategies for the treatment of USC. PLK1 is a type of serine/threonine kinase that is crucial for controlling the progression of the cell cycle, DNA damage response, and genome stability. Targeting PLK1 exhibits potent anti-tumorigenic activity in pre-clinical models of multiple cancer types, and several PLK1 inhibitors have shown significant clinical benefit and favorable safety profiles alone or in combination with other chemotherapeutic agents. Onvansertib is an oral, selective PLK1 inhibitor that exhibits anti-proliferative activity in multiple types of cancer cell and animal models and has demonstrated clinical activity and a favorable safety profile in recent clinical trials. Hence, we investigated the anti-tumorigenic effects of onvansertib in USC cell lines. Nanomolar concentrations of onvansertib significantly inhibited cellular proliferation, led to cell cycle G2 arrest, induced cellular stress and apoptosis, caused DNA damage, and reduced cell adhesion and invasion in ARK-1 and SPEC-2 cells. The combination of onvansertib with paclitaxel demonstrated a synergistic effect in cell proliferation inhibition via inducing cell apoptosis and DNA damage. Our results provide preclinical evidence that onvansertib may be an effective strategy to treat USC and deserves further evaluation in animal models and clinical trials.