AbstractNon-small-cell lung cancer (NSCLC) is a dominating type of lung cancer with high morbidity and mortality. Midazolam has been reported to promote cell apoptosis in NSCLC, but the molecular mechanism of midazolam remains to be further explored. In the current work, cell viability, proliferation, migration, and apoptosis rates of NSCLC cells treated with midazolam were measured using cell counting kit-8 assay, 5-ethynyl-2′-deoxyuridine (EdU) and colony formation assays, transwell, and flow cytometry assay, respectively, to evaluate the malignant behaviors. Western blot was applied to access EGFR/MEK/ERK pathway-related protein levels. The results demonstrated midazolam significantly declined the viability of NSCLC cells. Furthermore, midazolam restrained cell proliferation and migration and contributed to cell apoptosis in NSCLC. Midazolam exerted suppressive function to EGFR pathway during NSCLC development. Moreover, the activation of EGFR/MEK/ERK pathway abrogated the effects of midazolam on NSCLC cell proliferation, apoptosis, and migration. Taken together, midazolam exhibited anti-tumor effects hallmarked by EGFR pathway inhibition, providing a novel insight into the treatment of NSCLC.