The EGFR-TKIs (epidermal growth factor receptor-tyrosine kinases inhibitors) offer significant benefits to lung cancer patients with sensitive EGFR mutations; however, the development of acquired resistance poses a significant challenge and leads to poor prognosis. Thus, exploring novel therapeutic strategies to overcome EGFR-TKI resistance is urgently needed. This study introduces an innovative approach utilizing folic acid-modified milk exosomes loaded with c-kit siRNA (FA-mExo-siRNA-c-kit) to target EGFR-TKI resistance in lung cancer. Initially, gefitinib-resistant lung cancer cells exhibited stemness characteristics, including an epithelial-to-mesenchymal transition phenotype and elevated ABCG2 expression, which were closely regulated by c-kit. Subsequent treatment with FA-mExo-siRNA-c-kit demonstrated effective suppression of c-kit expression and attenuation of stemness traits in vitro, reducing gefitinib resistance. In xenograft and liver metastasis models, sequential administration of FA-mExo-siRNA-c-kit and gefitinib resulted in decreased tumor growth and prolonged survival. Mechanistically, c-kit was found to regulate the AKT/mTOR/4EBP1/eIF4E axis, promoting stemness and gefitinib resistance in lung cancer cells. This study unveils a novel mechanism of EGFR-TKI resistance involving the c-kit/mTOR pathway and proposes a promising therapeutic strategy for EGFR-TKI-resistant lung cancer, particularly with liver metastasis, using FA-mExo-siRNA-c-kit, suggesting potential for improved patient outcomes and warranting further investigation.© The author(s).