The GGGGCC (G4C2) repeat expansion in the C9orf72 gene is a common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but the mechanism is unknown. Using quantitative proteomics, the authors found that C9orf72 protein interacts with the actin-binding protein cofilin. Cofilin phosphorylation was increased in mouse motor neurons with reduced C9orf72 expression and in ALS patients with the C9orf72 G4C2 expansion. Moreover, dynamics were found to be disrupted in both cultured mouse motor neurons lacking C9orf72 and in induced pluripotent stem cell-derived motor neurons from people with ALS, suggesting that altered actin dynamics could contribute to the pathology underlying ALS and FTD.