<p>Drug resistance remains a major challenge in treating acute myeloid leukemia (AML), despite advancements in targeted therapies. We established cabozantinib-resistant FLT3-ITD⁺ AML cell lines (MV4-11-XR, Molm13-XR) from parental MV4-11 and Molm13 cells. In addition to resistance to cabozantinib, they also exhibited resistance to FDA-approved sorafenib and quizartinib with substantial increases in IC₅₀. The FLT3 D835Y mutation emerged in both cell lines, while an additional 1.3 kb deletion in <i>FLT3 (FLT3</i>¹^.³) was present in MV4-11-XR cells. Both resistant cells displayed higher proliferation rates and increased colony formation, as well as increased phosphorylation of FLT3 and its downstream signaling molecules, including ERK, STAT5, and AKT. Transcriptomic analysis identified 1113 and 1057 differentially expressed genes (DEGs) in MV4-11-XR and Molm13-XR, respectively, compared with their parentals, of which 81 and 74 DEGs are metabolic-related. Further metabolic assays confirmed that cabozantinib resistance was associated with significant metabolic alterations, including enhanced glycolysis with increased glucose uptake, lactate production, GAPDH activity, and glycolytic gene expression, as well as impaired oxidative phosphorylation and reduced mitochondria mass. Further in silico drug screening and in vitro experiments demonstrated that PI3K/mTOR dual inhibitor omipalisib and HSP90 inhibitor radicicol effectively reversed the metabolic reprogramming in cabozantinib-resistant cells. Moreover, both omipalisib and radicicol exhibited synergistic effects with cabozantinib, highlighting their therapeutic potential. Overall, we identified metabolic dysregulation as a hallmark of cabozantinib resistance and suggested that targeting metabolic vulnerabilities with PI3K/mTOR or HSP90 inhibitors could be an option to mitigate drug resistance.</p><p></p>