A hexanucleotide GGGGCC repeat expansion in the noncoding region of the C9ORF72 gene is the most common genetic abnormality in familial and sporadic amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The function of the C9ORF72 protein is unknown, as is the mechanism by which the repeat expansion could cause disease. Induced pluripotent stem cell (iPSC)-differentiated neurons from C9ORF72 ALS patients revealed disease-specific (1) intranuclear GGGGCCexp RNA foci, (2) dysregulated gene expression, (3) sequestration of GGGGCCexp RNA binding protein ADARB2, and (4) susceptibility to excitotoxicity. These pathological and pathogenic characteristics were confirmed in ALS brain and were mitigated with antisense oligonucleotide (ASO) therapeutics to the C9ORF72 transcript or repeat expansion despite the presence of repeat-associated non-ATG translation (RAN) products. These data indicate a toxic RNA gain-of-function mechanism as a cause of C9ORF72 ALS and provide candidate antisense therapeutics and candidate human pharmacodynamic markers for therapy. •iPSC neurons mirror dysregulated gene expression in C9ORF72 brain•Aggregated RNA binding proteins contribute to toxicity•C9ORF72 iPSC neurons are highly susceptible to excitotoxicity.•Pathology and toxicity is abrogated with antisense to C9ORF72 expansion Donnelly et al. provide evidence for RNA toxicity, through the use of induced pluripotent cells, as the cause of amyotrophic lateral sclerosis and frontotemporal dementia in patients with mutation in the C9ORF72 gene and a cellular rescue using antisense oligonucleotide therapy.