A GGGGCC repeat expansion in the C9ORF72 gene has been identified as the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. The repeat expansion undergoes unconventional translation to produce dipeptide repeat proteins. Although it has been reported that dipeptide repeat proteins cause neurotoxicity, the underlying mechanism has not been fully elucidated. In this study, we show that the expression of proline-arginine repeat protein (poly-PR) reduces levels of ribosomal RNA and causes neurotoxicity. The poly-PR-induced neurotoxicity is restored by the acceleration of ribosomal RNA synthesis. This result suggests that the poly-PR-induced inhibition of ribosome biogenesis contributes to the poly-PR-induced neurotoxicity. Furthermore, we show that poly-PR interacts with multiple DEAD-box RNA helicases and inhibits the function of at least one of them, and that the reduction in the levels of some RNA helicases results in both the decrease in ribosomal RNA levels and the increase in neuronal cell death. Altogether, these results suggest that poly-PR causes neuronal toxicity by inhibiting the DEAD-box RNA helicase-mediated ribosome biogenesis.