Demethyleneberberine (DMB) is a natural active component of medicinal plant Cortex phellodendri chinensis with favorable bioactivity. However, the role of DMB in suppressing non-small cell lung cancer (NSCLC) remains unknown.In this study, we aimed to examine the effect and underlying mechanism of DMB in suppressing NSCLC.CCK8 assay and colony formation assay were utilized to assess the efficiency of DMB on the viability and colony formation capacity of NSCLC cells. Flow cytometry and β-Galactosidase Staining Kit were utilized to determine the efficiency of DMB on the cell cycle and cellular senescence of NSCLC cells. RT-qPCR and Western blot were used to detect the effect of DMB on cell cycle and cellular senescence related gene and protein expression of NSCLC cells. In vivo tumor model was established to evaluate the anti NSCLC effect of DMB. In addition, RNA-seq analysis was performed to detect the differential gene expression after DMB treatments.In this study, we revealed that DMB exhibits efficient inhibitory effect on NSCLC cell proliferation and tumor xenografts growth in vivo. We also demonstrated that DMB could inhibit cell migration by suppressing epithelial-mesenchymal transition (EMT) and trigger cell cycle arrest by down-regulating the expression of cell cycle related genes in NSCLC cells. In addition, DMB treatment efficiently induces cellular senescence of NSCLC cells. From the RNA-seq analysis, we found that DMB accelerates senescence through suppressing HIF-1α expression, which was further elucidated by overexpressing HIF-1α in NSCLC to reduce the inhibitory effect of DMB. Furthermore, we also revealed that DMB decreases the expression of c-Myc, an up-stream protein of HIF-1α.Taken together, we first report that DMB inhibits NSCLC progress through inducing cell cycle arrest and triggering cellular senescence by downregulating c-Myc/HIF-1α pathway.Copyright © 2021 Elsevier GmbH. All rights reserved.