Abstract: Approximately 85% of all lung cancer cases are classified as non-small cell lung cancer (NSCLC). Given its poor prognosis and resistance to radiotherapy and chemotherapy, there is an urgent need to elucidate its molecular mechanisms to develop novel and more effective therapeutic strategies. In prior research, we identified nobiletin from a compound library and confirmed it as a novel natural BH3 mimetic. Nobiletin synergized with vorinostat to induce autophagy and apoptosis in small-cell lung cancer. In the current study, we further demonstrate that nobiletin, either alone or in combination with vorinostat, exerts inhibitory effects on NSCLC. Specifically, the combination of nobiletin and vorinostat suppressed the proliferation of NSCLC A549 cells. Nobiletin, used alone or with vorinostat, induced apoptosis in A549 cells by mimicking BH3-only proteins, which included down-regulating anti-apoptotic proteins such as B-cell lymphoma-2 (BCL-2) and MCL-1, up-regulating apoptosis-related proteins Cleaved-Caspase-3 and Cleaved-PARP, and increasing BH3-only protein expression. Nobiletin binding to BCL-2 facilitated the dissociation of the Beclin-1/BCL-2 complex, thereby elevating levels of free Beclin-1. Furthermore, the combination of nobiletin and vorinostat enhanced the expression of LC3A/BII and forkhead box O1 (FOXO1), ultimately inducing autophagy in A549 cells. Eukaryotic transcriptome sequencing revealed that the combination treatment primarily inhibits tumor cell proliferation by modulating TRKC protein expression and suppressing phosphorylation of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway. Therefore, our results indicate that nobiletin, a natural BH3 mimetic, synergizes with vorinostat to regulate both apoptosis and autophagy in NSCLC.