Ataxin-3 is a member of the polyglutamine family of proteins, which are associated with at least nine different neurodegenerative diseases. In the disease state, expansion of the polyglutamine tract leads to dysfunction and death of neurons, as well as formation of proteinaceous aggregates known as nuclear inclusions. Intriguingly, both expanded and non-expanded forms of ataxin-3 are observed within these nuclear inclusions. Ataxin-3 is the smallest of the polyglutamine disease proteins and in its expanded form causes the neurodegenerative disorder Machado-Joseph disease. Using a non-pathological variant containing 28 residues in its polyglutamine tract, we have probed the folding and misfolding pathways of ataxin-3. We describe here the first equilibrium folding pathway delineated for any polyglutamine protein and show that ataxin-3 folds reversibly via a single intermediate species. We have also explored further the misfolding potential of the protein and found that partial destabilization of ataxin-3 by chemical denaturation leads to the formation of fibrillar aggregates by the non-pathological variant. These results provide an insight into the possible mechanisms by which polyglutamine expansion may affect the stability and conformation of the protein. The implications of this are considered in the wider context of the development and pathogenesis of polyglutamine diseases.