Recognition of viral dsRNA by PKR (protein kinase RNA-activated) causes its autophosphorylation and phosphorylation of eIF2α (eukaryotic translation initiation factor 2 alpha), leading to global translational inhibition. Increasing evidence shows that PKR can also be phosphorylated during the cell cycle and under stress conditions without infection. To identify the endogenous dsRNAs that can activate PKR under these conditions, Kim et al. utilized a formaldehyde-mediated cross-linking and immunoprecipitation sequencing (fCLIP-seq) approach to profile PKR-interacting RNAs in S- or M-phase-arrested HeLa cells. They found that the major class of PKR-interacting dsRNAs was derived from mitochondrial RNAs (mtRNAs). Decreasing the amount of mtRNAs by knocking down POLRMT, a mitochondrial RNA polymerase, reduced the level of phosphorylated PKR (pPKR) and eIF2α. Redirecting PKR into the mitochondrial matrix only increased the level of pPKR, suggesting that mitochondrial export of pPKR is required for eIF2α phosphorylation. During M phase and under stress conditions, the upregulation of mtRNAs and/or cytosolic release of mtRNAs results in increased mtRNA–PKR duplex formation and PKR activation. These results show that mtRNAs can induce PKR activation and provide new insight into the role of mtRNA in translational regulation.