The precise mechanism underlying the conversion of normal prion protein (PrPC) into abnormal prion protein (PrPSc) remains unclear. Protein misfolding cyclic amplification (PMCA), an in vitro technique used for amplifying PrPSc, results in PrPSc replication that preserves the strain-specific characteristics of the input PrPSc; thus, PMCA mimics the process of in vivo PrPSc replication. Previous work has demonstrated that in PMCA, nucleic acids are critical for PrPSc amplification, but little information has been reported on glycosaminoglycan (GAG) participation in PrPSc replication in vitro. Here, we investigated whether GAGs play a role in the faithful replication of PrPSc by using a modified PMCA performed with baculovirus-derived recombinant PrP (Bac-PrP) as a substrate. The addition of heparan sulfate (HS) or its analog heparin (HP) restored the conversion efficiency in PMCA that was inhibited through nucleic acid depletion. Moreover, the PMCA products obtained under these conditions were infectious and preserved the properties of the input PrPSc. These data suggest that HS and HP play the same role as nucleic acids in facilitating faithful replication of prions in PMCA. Furthermore, we showed that HP binds to both Bac-PrP and Bac-PrPSc through the sulfated groups present on HP and that the N-terminal domain of Bac-PrPSc might potentially not be involved in the binding to HP. These results suggest that the interaction of GAGs such as HS and HP with PrPC and/or PrPSc through their sulfate groups is critical for the faithful replication of prions.