Significance The small, lipid-binding protein, α-synuclein (α-syn), is associated with neurodegenerative diseases. α-Syn exists in various splice isoforms, and isoform expression varies with disease, but how these isoforms affect protein function is unknown. Using a yeast model expressing α-syn splice variants, we show that inhibition of sterol synthesis differentially affects α-syn plasma membrane association, vesicular association, and cellular toxicity, depending on which α-syn isoform is expressed. This result suggests that higher membrane sterol concentrations may be protective of synucleinopathy progression. Given the common use of cholesterol-reducing statins and their potential effects on membrane-binding proteins, further investigation of how sterol concentration and α-syn splice isoforms affect vesicular trafficking in synucleinopathies is warranted.