The molecular interaction between PrP and 14-3-3 beta and the possible interactional domain between two proteins were studied by co-immunoprecipitation, pull down and FRET assays. The results showed that PrP protein could interact with 14-3-3 beta in vitro and in vivo. The domain which responded for the interaction was located at C-terminal of PrP (amino acid residues 106 to 126). This study of the interaction between PrP and 14-3-3 protein further provided the insight into the potential role of 14-3-3 in the biological function of PrP and the pathogenesis of prion disease.