We used a bovine brain cDNA library to perform a yeast two-hybrid assay with bovine mature PrP(C) as bait. The screening result showed that alphaB-crystalline interacted with PrP(C). The interaction was further evaluated both in vivo and in vitro with different methods, such as immunofluorescent colocalization, native polyacrylamide-gel electrophoresis, and IAsys biosensor assays. The results suggested that alphaB-crystalline may have the ability to refold denatured prion proteins, and provided first evidence that alphaB-crystalline is directly associated with prion protein.