Prion or PrP^Sc is a proteinaceous infectious agent that consists of a misfolded and aggregated form of a sialoglycoprotein called prion protein or PrP^C. PrP^C has two sialylated N-linked carbohydrates. In PrP^Sc, the glycans are directed outward, with the terminal sialic acid residues creating a negative charge on the surface of prion particles. The current study proposes a new hypothesis that electrostatic repulsion between sialic residues creates structural constraints that control prion replication and PrP^Sc glycoform ratio. In support of this hypothesis, here we show that diglycosylated PrP^C molecules that have more sialic groups per molecule than monoglycosylated PrP^C were preferentially excluded from conversion. However, when partially desialylated PrP^C was used as a substrate, recruitment of three glycoforms into PrP^Sc was found to be proportional to their respective populations in the substrate. In addition, hypersialylated molecules were also excluded from conversion in the strains with the strongest structural constraints, a strategy that helped reduce electrostatic repulsion. Moreover, as predicted by the hypothesis, partial desialylation of PrP^C significantly increased the replication rate. This study illustrates that sialylation of N-linked glycans creates a prion replication barrier that controls replication rate and glycoform ratios and has broad implications.