In prion diseases, the cellular prion protein (PrP^C) becomes misfolded into the pathogenic scrapie isoform (PrP^Sc) responsible for prion infectivity. We show here that peptides derived from the prion protein N terminus have potent antiprion effects. These peptides are composed of a hydrophobic sequence followed by a basic segment. They are known to have cell-penetrating ability like regular cell-penetrating peptides (CPPs), short peptides that can penetrate cellular membranes. Healthy (GT1–1) and scrapie-infected (ScGT1–1) mouse neuronal hypothalamic cells were treated with various CPPs, including the prion protein-derived CPPs. Lysates were analyzed for altered protein levels of PrP^C or PrP^Sc. Treatment with the prion protein-derived CPPs mouse mPrP_1–28 or bovine bPrP_1–30 significantly reduced PrP^Sc levels in prion-infected cells but had no effect on PrP^C levels in noninfected cells. Further, presence of prion protein-derived CPPs significantly prolonged the time before infection was manifested when infecting GT1–1 cells with scrapie. Treatment with other CPPs (penetratin, transportan-10, or poly-L-arginine) or prion protein-derived peptides lacking CPP function (mPrP_23–28, mPrP_19–30, or mPrP_23–50) had no effect on PrP^Sc levels. The results suggest a mechanism by which the signal sequence guides the prion protein-derived CPP into a cellular compartment, where the basic segment binds specifically to PrP^Sc and disables formation of prions.—Löfgren, K., Wahlström, A., Lundberg, P., Langel, U., Gräslund, A., and Bedecs, K. Antiprion properties of prion protein-derived cell-penetrating peptides.