As an approved targeting drug, crizotinib has been widely used in the treatment of patients with non-small cell lung cancer (NSCLC) with anaplastic lymphoma kinase (ALK) rearrangements or c-ros oncogene 1 (ROS1) fusions and has demonstrated remarkable therapeutic effects. However, crizotinib-treated patients frequently experience drug resistance, and there are still some underlying mechanisms, which remain unclear. Autophagy, a cellular process that involves the degradation and recycling of cellular components, has been implicated in the development of drug resistance. In this study, we aim to elucidate the mechanisms of crizotinib resistance involving autophagy dysregulation and identify novel therapeutic targets to overcome this resistance.We first established a model for crizotinib resistance in HCC78 and H3122 cells. Next, the level of proliferation, apoptosis, autophagy flux, and reactive oxygen species (ROS) of these cells were measured. Subsequently, we analyzed the published single-cell RNA sequencing data from three ALK-rearranged lung cancer organoid samples and performed a metabolomics assay on crizotinib-resistant HCC78 cells. Finally, the therapeutic effects were confirmed in vitro by targeting autophagy flux.Crizotinib induced cell apoptosis and growth arrest by promoting the accumulation of autophagosomes through the inhibition of autophagy flux in ROS1 + or ALK + NSCLC. In contrast, crizotinib-resistant NSCLC cells showed inactivation of signal transducer and activator of transcription 3 (STAT3) phosphorylation and downregulation of prostaglandin endoperoxide synthase 2 (PTGS2), leading to an increase in the metabolite arachidonic acid (AA). AA further promoted autophagy flux and reduced autophagosome accumulation, driving crizotinib resistance under conditions of drug stress. Moreover, chloroquine (CQ), anti-malaria drug and lysosome inhibitor developed in 1940, could induce cell death in crizotinib-resistant NSCLC by blocking AA-mediated autophagy flux and facilitating autophagosome accumulation, significantly enhancing the treatment efficacy of crizotinib in drug-resistant NSCLC.We discovered a new mechanism of first generation ALK- and ROS1-TKIs resistance, which points to the role of the metabolite AA in resistance to tyrosine kinase inhibitors. It may potentially provide an alternative strategy to overcoming crizotinib resistance in NSCLC treatment by reversing AA-mediated autophagy.Copyright © 2025 AME Publishing Company. All rights reserved.