Cancer cells depend on mitochondria to sustain their increased metabolic need and mitochondria therefore constitute possible targets for cancer treatment. We recently developed small‐molecule inhibitors of mitochondrial transcription (IMTs) that selectively impair mitochondrial gene expression. IMTs have potent antitumor properties in vitro and in vivo, without affecting normal tissues. Because therapy‐induced resistance is a major constraint to successful cancer therapy, we investigated mechanisms conferring resistance to IMTs. We employed a CRISPR‐Cas9 (clustered regularly interspaced short palindromic repeats)‐(CRISP‐associated protein 9) whole‐genome screen to determine pathways conferring resistance to acute IMT1 treatment. Loss of genes belonging to von Hippel–Lindau (VHL) and mammalian target of rapamycin complex 1 (mTORC1) pathways caused resistance to acute IMT1 treatment and the relevance of these pathways was confirmed by chemical modulation. We also generated cells resistant to chronic IMT treatment to understand responses to persistent mitochondrial gene expression impairment. We report that IMT1‐acquired resistance occurs through a compensatory increase of mitochondrial DNA (mtDNA) expression and cellular metabolites. We found that mitochondrial transcription factor A (TFAM) downregulation and inhibition of mitochondrial translation impaired survival of resistant cells. The identified susceptibility and resistance mechanisms to IMTs may be relevant for different types of mitochondria‐targeted therapies. Synopsis A CRISPR‐Cas9 screen shows that mTORC1 loss increases OXPHOS and prevents IMT1‐induced cell death whereas loss of VHL confers resistance in an OXPHOS‐independent fashion. A CRISPR‐Cas9 screen shows that loss of mTORC1 or VHL cause resistance to the inhibition of mtDNA transcription in cancer cells. Rapamycin promotes survival of IMT1‐treated cancer cells by enhancing mtDNA expression and OXPHOS. Cells with chronic IMT1 resistance have increased mtDNA levels and OXPHOS capacity. Decreased mtDNA levels or impaired mitochondrial translation sensitize cancer cells to IMT1 treatment. A CRISPR‐Cas9 screen shows that mTORC1 loss increases OXPHOS and prevents IMT1‐induced cell death whereas loss of VHL confers resistance in an OXPHOS‐independent fashion.