We previously reported that autophagy is upregulated in Prnp-deficient (Prnp ( 0/0) ) hippocampal neuronal cells in comparison to cellular prion protein (PrP (C) )-expressing (Prnp (+/+) ) control cells under conditions of serum deprivation. In this study, we determined whether a protective mechanism of PrP (C) is associated with autophagy using Prnp ( 0/0) hippocampal neuronal cells under hydrogen peroxide (H 2O 2)-induced oxidative stress. We found that Prnp ( 0/0) cells were more susceptible to oxidative stress than Prnp (+/+) cells in a dose- and time-dependent manner. In addition, we observed enhanced autophagy by immunoblotting, which detected the conversion of microtubule-associated protein 1 light chain 3 β (LC3B)-I to LC3B-II, and we observed increased punctate LC3B immunostaining in H 2O 2-treated Prnp ( 0/0) cells compared with H 2O 2-treated control cells. Interestingly, this enhanced autophagy was due to impaired autophagic flux in the H 2O 2-treated Prnp ( 0/0) cells, while the H 2O 2-treated Prnp (+/+) cells showed enhanced autophagic flux. Furthermore, caspase-dependent and independent apoptosis was observed when both cell lines were exposed to H 2O 2. Moreover, the inhibition of autophagosome formation by Atg7 siRNA revealed that increased autophagic flux in Prnp (+/+) cells contributes to the prosurvival effect of autophagy against H 2O 2 cytotoxicity. Taken together, our results provide the first experimental evidence that the deficiency of PrP (C) may impair autophagic flux via H 2O 2-induced oxidative stress.