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Abstract Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) provide clinical benefits over chemotherapy for lung cancer patients with EGFR activating mutations. Despite initial clinical responses, long‐term efficacy is not possible because of acquired resistance to these therapies. We have developed EGFR TKI drug‐tolerant (DT) human lung cancer cell lines as a model for de novo resistance. Mass spectroscopic analysis revealed that the cytochrome P450 protein, CYP51A1 (Lanosterol 14α‐demethylase), which is directly involved with cholesterol synthesis, was significantly upregulated in the DT cells. Total cellular cholesterol, and more specifically, mitochondrial cholesterol, were found to be upregulated in DT cells. We then used the CYP51A1 inhibitor, ketoconazole, to downregulate cholesterol synthesis. In both parental and DT cells, ketoconazole and EGFR TKIs acted synergistically to induce apoptosis and overcome the development of EGFR tolerance. Lastly, this combination therapy was shown to shrink the growth of tumors in an in vivo mouse model of EGFR TKI resistance. Thus, our study demonstrates for the first time that ketoconazole treatment inhibits upregulation of mitochondrial cholesterol and thereby overcomes EGFR‐TKI resistance in lung cancer cells.