The cellular prion protein (PrP(c)) is a glycosyl-phosphatidylinositol (GPI)-anchored protein trafficking in the secretory and endocytic pathway and localized mainly at the plasma membrane. Conversion of PrP(c) into its pathogenic isoform PrP(Sc) is associated with pathogenesis and transmission of prion diseases. Intramolecular cleavage in the middle, the extreme C-terminal part or within the GPI anchor and shedding of PrP(c) modulate this conversion process by reducing the substrate for prion formation. These phenomena provide similarities with the processing of amyloid precursor protein in Alzheimer's disease. Sorting nexins are a family of proteins with important functions in protein trafficking. In this study, we investigated the role of the newly described sorting nexin 33 (SNX33) in trafficking and processing of PrP(c). We found that overexpression of SNX33 in neuronal and non-neuronal cell lines resulted in increased shedding of full-length PrP(c) from the plasma membrane and modulated the rate of PrP(c) endocytosis. This was paralleled by reduction of PrP(Sc) formation in persistently and newly infected cells. Using deletion mutants, we demonstrate that production of PrP fragment N1 is not influenced by SNX33. Our data provide new insights into the cellular mechanisms of PrP(c) shedding and show how this can affect cellular PrP(Sc) conversion.