Polyglutamine (polyQ) diseases are classified as conformational neurodegenerative diseases, like Alzheimer and Parkinson diseases, and they are caused by proteins with an abnormally expanded polyQ stretch. However, conformational changes of the expanded polyQ protein and the toxic conformers formed during aggregation have remained poorly understood despite their important role in pathogenesis. Here we show that a β-sheet conformational transition of the expanded polyQ protein monomer precedes its assembly into β-sheet–rich amyloid-like fibrils. Microinjection of the various polyQ protein conformers into cultured cells revealed that the soluble β-sheet monomer causes cytotoxicity. The polyQ-binding peptide QBP1 prevents the toxic β-sheet conformational transition of the expanded polyQ protein monomer. We conclude that the toxic conformational transition, and not simply the aggregation process itself, is a therapeutic target for polyQ diseases and possibly for conformational diseases in general.