Eg5, also known as KIF11, is a motor protein essential for establishing a bipolar spindle and ensuring proper chromosome congression during mitosis. It is amplified in various human cancers and serves as a critical oncogene driving tumour progression. However, the role and clinical significance of Eg5 in gastric cancer has remained elusive. In this study, we showed that Eg5 is upregulation in gastric cancer tissues and is negatively associated with patient prognosis. The ablation of Eg5 inhibits the proliferation and migration of gastric cancer cells and suppresses tumour growth in xenograft mice. Mechanistically, Eg5 ablation induces the formation of monopolar spindle, leading to cell apoptosis and consequent inhibition of tumour growth. Furthermore, Arry520 is demonstrated as a potent Eg5 inhibitor which blocks tumour growth by increasing the formation of cell monopolar spindle and inducing apoptosis. Arry520 exhibits efficiently therapeutic effects on gastric cancer in tumour organoid models, cell-derived xenografts and patient-derived xenografts (PDX) in mice. Collectively, our findings provide evidence for the oncogenic properties of the mitotic protein Eg5 and identify Arry520 as a promising strategy to combat gastric cancer.Copyright © 2025 The Author(s). Published by Elsevier B.V. All rights reserved.