GGGGCC (G4C2) hexanucleotide repeat expansion in theC9ORF72gene is the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). The repeat is bidirectionally transcribed and confers gain of toxicity. However, the underlying toxic species is debated, and it is not clear whether antisense CCCCGG (C4G2) repeat expanded RNAs contribute to disease pathogenesis. Our study shows thatC9ORF72antisense C4G2repeat expanded RNAs trigger the activation of the PKR/eIF2α-dependent integrated stress response independent of dipeptide repeat proteins that are produced through repeat-associated non-AUG-initiated translation, leading to global translation inhibition and stress granule formation. Reducing PKR levels with either siRNA or morpholinos mitigates integrated stress response and toxicity caused by the antisense C4G2RNAs in cell lines, primary neurons, and zebrafish. Increased phosphorylation of PKR/eIF2α is also observed in the frontal cortex ofC9ORF72FTD/ALS patients. Finally, only antisense C4G2, but not sense G4C2, repeat expanded RNAs robustly activate the PKR/eIF2α pathway and induce aberrant stress granule formation. These results provide a mechanism by which antisense C4G2repeat expanded RNAs elicit neuronal toxicity in FTD/ALS caused byC9ORF72repeat expansions.