Transmissible spongiform encephalopathies are lethal neurodegenerative disorders that present with aggregated forms of the cellular prion protein (PrP^C), which are known as PrP^Sc. Prions from different species vary considerably in their transmissibility to xenogeneic hosts. The variable transmission barriers depend on sequence differences between incoming PrP^Sc and host PrP^C and additionally, on strain-dependent conformational properties of PrP^Sc. The β₂-α₂ loop region within PrP^C varies substantially between species, with its structure being influenced by the residue types in the 2 amino acid sequence positions 170 (most commonly S or N) and 174 (N or T). In this study, we inoculated prions from 5 different species into transgenic mice expressing either disordered-loop or rigid-loop PrP^C variants. Similar β₂-α₂ loop structures correlated with efficient transmission, whereas dissimilar loops correlated with strong transmission barriers. We then classified literature data on cross-species transmission according to the 170S/N polymorphism. Transmission barriers were generally low between species with the same amino acid residue in position 170 and high between those with different residues. These findings point to a triggering role of the local β₂-α₂ loop structure for prion transmissibility between different species.