Objective Prion diseases are infectious, lethal neurodegenerative disorders principally caused by the conformational conversion of prion protein (PrP) from its cellular form (PrP^C) into a protease-resistant, aggregated form (PrP^Sc) in humans and various vertebrate species. We have recently reported a cryo-EM structure of an amyloid fibril formed by full-length human PrP, which features a parallel in-register intermolecular β sheet architecture. However, it is unclear whether amyloid fibrils from full-length human PrP^C are cytotoxic and transmissible.Methods Sarkosyl-insoluble Western blotting and cell viability assays were used to detect PrP aggregation and cell viability, respectively. Oxidative stress detection and annexin V-FITC apoptosis detection assays were also used for the determination of ROS production and cell apoptosis, respectively.Results Human PrP fibrils are cytotoxic, and transmissible to induce the misfolding of endogenous PrP^C not only in cells but also in the frontal cortices of infant mice. The PrP fibrils also induce severe mitochondrial damage in cells stably expressing PrP^C. Importantly, the PrP fibrils elevate ROS production via aggravating mitochondrial stress resulting from PrP aggregation and induce severe late apoptosis in cells stably expressing PrP^C.Conclusion We demonstrate that PrP fibrils prepared in vitro are cytotoxicity and have pathogenic potential.