--- - |2 This study aims to investigate the effect of polo‐like kinase 1 (PLK1) inhibition on cisplatin (DDP)‐resistant gastric cancer (GC) cells. Together, our experimental results illustrated that the DDP resistance of GC cells might be associated with the aberrant overexpression of PLK1. PLK1 inhibition, including si‐PLK1 and BI2536 treatment, could restore the chemosensitivity of drug‐resistant SGC‐7901/DDP cells and enhance the efficacy of DDP, revealing the potential value of PLK1 inhibition in GC chemotherapy. Abstract Objective This study aims to investigate the effect of polo‐like kinase 1 (PLK1) inhibition on cisplatin (DDP)‐resistant gastric cancer (GC) cells. Methods: The transcriptional level of PLK1 was measured by quantitative reverse‐transcription polymerase chain reaction. Expressions of PLK1 and its downstream mediators as well as autophagy‐related protein LC3 I/LC3 II were detected by western blot. An 3‐(4,5‐Dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT) assay and 5‐ethynyl‐2′‐deoxyuridine immunofluorescent staining were conducted to evaluate the cell viability and replication activity separately. Flow cytometry was carried out to determine the cell cycle status. The GFP‐LC3 vector contributed toward tracking the formation and aggregation of autophagosomes. Results: Drug‐resistant SGC‐7901/DDP cells showed insignificant changes in all phases after DDP treatment, including DNA replication, cell proliferation, cell cycle, and apoptosis, whereas DDP could significantly improve the autophagy level of SGC‐7901/DDP as well as PLK1expression. By downregulating the expression of PLK1, both BI2536 andsi‐PLK1 enhanced SGC‐7901/DDP sensitivity to DDP, suppressing the proliferation and autophagy as well as improving the apoptosis rate. PLK1 inhibition also resulted in the repression of cell division regulators CDC25C and cyclin B1. Conclusion: Together, our experimental results illustrated that the DDP resistance of GC cells might be associated with the aberrant overexpression of PLK1. PLK1 inhibition, including si‐PLK1 and BI2536 treatment, could restore the chemosensitivity of drug‐resistant SGC‐7901/DDP cells and enhance the efficacy of DDP, revealing the potential value of PLK1 inhibition in GC chemotherapy. - 'Journal of Cellular Physiology, EarlyView. '