Polo-like kinase 1 (PLK1) is crucial in regulating cell division and has been shown to have an oncogenic function in several cancers. Since PLK1 overexpression is closely related to tumorigenesis and has been correlated with poor clinical outcomes, specific inhibition of PLK1 in cancer cells is a promising approach for developing new anticancer drugs. In this context, the aim of the present study was to evaluated the potential cytotoxic effects of GSK461364A, a competitive inhibitor for PLK1, in gastric cancer cell line SNU-1 cells and explored its cytotoxic mechanism. The cells were exposed to GSK461364A at different concentrations ranging from 1 to 40 μM for 24 h, and it showed considerable cytotoxicity with an IC50 value of 4.34 μM. The treatment of SNU-1 cells with GSK461364A results in cell cycle arrest at the G2/M phase, decreased mitochondrial membrane potential, and increased apoptosis as indicated by Annexin V binding assay. In addition, GSK461364A treatment significantly increased the total oxidant (TOS) level, a signal of oxidative stress, and increased cleaved PARP and 8-oxo-dG levels as an indicator of DNA damage. ELISA experiments evaluating Bax, BCL-2, and cleaved caspase 3 also confirmed the apoptotic effect of GSK461364A. Current findings suggest that GSK461364A may be a chemotherapeutic agent in patients with gastric cancer. Nevertheless, more research is needed to evaluate GSK461364A as a cancer treatment drug.