Prion diseases are fatal and infectious neurodegenerative disorders characterized by the accumulation of an abnormally folded form of the prion protein (PrP), termed PrP(Sc). Prion replication triggers endoplasmic reticulum (ER) stress, neuronal dysfunction, and apoptosis. In this study we analyze the effect of perturbations in ER homeostasis on PrP biochemical properties and prion replication. ER stress led to the generation of a mis-folded PrP isoform, which is detergent-insoluble and protease-sensitive. To understand the mechanism by which ER stress generates PrP misfolding, we assessed the contribution of different signaling pathways implicated in the unfolded protein response. Expression of a dominant negative form of IRE1 alpha or XBP-1 significantly increased PrP aggregation, whereas overexpression of ATF4 or an active mutant form of XBP-1 and ATF6 had the opposite affect. Analysis of prion replication in vitro revealed that the PrP isoform generated after ER stress is more efficiently converted into PrP(Sc) compared with the protein extracted from untreated cells. These findings indicate that ER-damaged cells might be more susceptible to prion replication. Because PrP(Sc) induces ER stress, our data point to a vicious cycle accelerating prion replication, which may explain the rapid progression of the disease.