Conformational conversion of normal cellular prion protein PrP^C into pathogenic PrP^Sc is central to the pathogenesis of prion diseases. However, the pathogenic mechanism remains unknown. Here we show that post-Golgi vesicular trafficking is significantly delayed in prion-infected N2a cells. Accordingly, cell surface expression of membrane proteins examined, including PrP^C, insulin receptor involved in neuroprotection, and attractin, whose mutation causes prion disease-like spongiform neurodegeneration, is reduced. Instead, they accumulate in the Golgi apparatus. PrP^Sc is detected throughout endosomal compartments, being particularly abundant in recycling endosome. We also show reduced surface expression of PrP^C and insulin receptor in prion-infected mouse brains well before the onset of disease. These results suggest that prion infection might impair post-Golgi trafficking of membrane proteins to the cell surface in neurons via PrP^Sc accumulated in recycling endosome, and eventually induce neuronal dysfunctions associated with prion diseases.