To investigate the effects of PLK1 inhibitors on Osimertinib-resistant NCI-H1975 cell lines and the anti-tumor effect combined with Osimertinib. Osimertinib resistant NCI-H1975 cell lines were constructed by the methods of concentration-increasing.; The classical tumor pathway inhibitor library compounds were combined with Osimertinib on Osimertinib-resistant cells to screen compounds with synergistic effects with Osimertinib; The gene set enrichment analysis (GSEA) was used to investigate the activations of signaling pathways in Osimertinib-resistant cells; Sulforhodamine B (SRB) staining was used to investigate the inhibitory effect of PLK1 inhibitors on Osimertinib-resistant cells and the anti-tumor effect of PLK1 inhibitors combined with Osimertinib. Osimertinib-resistant cells lines H1975-resistance (resistance index=43.45) were successfully established. The PLK1 inhibitors BI 2536 and GSK461324 have synergistic effect with Osimertinib. Compared with Osimertinib-sensitive cells, PLK1 regulatory pathway and cell cycle pathway were significantly activated in Osimertinib-resistant cells. In a cohort of NSCLC patients with epidermal growth factor receptor mutations treated with Osimertinib, PLK1 mRNA levels were negatively correlated with progression free survival of patients (R=0.62, P<0.05), confirming that excessive activation of PLK1 in NSCLC cells may cause cell resistance to Osimertinib. Further vitro experiments showed that, The IC50 of PLK1 inhibitors BI 6727 and GSK461324 in Osimertinib-resistant cells were lower than those in sensitive ones. Compared to the mono treatment of Osimertinib, PLK1 inhibitors combined with Osimertinib behave significantly stronger effect on the proliferation of Osimertinib-resistant cells. PLK1 inhibitors combined with Osimertinib behave stronger anti-tumor effect to Osimertinib-resistant NSCLC cells and may be used for intervention and treatment of Osimertinib resistance.