A genome-wide CRISPR interference screen shows that a signalling pathway involving OMA1, DELE1 and the eIF2 alpha kinase HRI relays mitochondrial stress to the cytosol to trigger the integrated stress response. In mammalian cells, mitochondrial dysfunction triggers the integrated stress response, in which the phosphorylation of eukaryotic translation initiation factor 2 alpha (eIF2 alpha) results in the induction of the transcription factor ATF4(1-3). However, how mitochondrial stress is relayed to ATF4 is unknown. Here we show that HRI is the eIF2 alpha kinase that is necessary and sufficient for this relay. In a genome-wide CRISPR interference screen, we identified factors upstream of HRI: OMA1, a mitochondrial stress-activated protease; and DELE1, a little-characterized protein that we found was associated with the inner mitochondrial membrane. Mitochondrial stress stimulates OMA1-dependent cleavage of DELE1 and leads to the accumulation of DELE1 in the cytosol, where it interacts with HRI and activates the eIF2 alpha kinase activity of HRI. In addition, DELE1 is required for ATF4 translation downstream of eIF2 alpha phosphorylation. Blockade of the OMA1-DELE1-HRI pathway triggers an alternative response in which specific molecular chaperones are induced. The OMA1-DELE1-HRI pathway therefore represents a potential therapeutic target that could enable fine-tuning of the integrated stress response for beneficial outcomes in diseases that involve mitochondrial dysfunction.