Previously, we have shown the loss of anti-Bax function in Creutzfeldt Jakob disease (CJD)-associated prion protein (PrP) mutants that are unable to generate cytosolic PrP (CyPrP). To determine if the anti-Bax function of PrP modulates the manifestation of prion diseases, we further investigated the anti-Bax function of eight familial Gerstmann-Sträussler-Scheinker Syndrome (GSS)-associated PrP mutants. These PrP mutants contained their respective methionine ((M)) or valine ((V)) at codon 129. All of the mutants lost their ability to prevent Bax-mediated chromatin condensation or DNA fragmentation in primary human neurons. In the breast carcinoma MCF-7 cells, the F198S(V), D202N(V), P102L(V) and Q217R(V) retained, whereas the P102L(M), P105L(V), Y145stop(M) and Q212P(M) PrP mutants lost their ability to inhibit Bax-mediated condensed chromatin. The inhibition of Bax-mediated condensed chromatin depended on the ability of the mutants to generate cytosolic PrP. However, except for the P102L(V), none of the mutants significantly inhibited Bax-mediated caspase activation. These results show that the cytosolic PrP generated from the GSS mutants is not as efficient as wild type PrP in inhibiting Bax-mediated cell death. Furthermore, these results indicate that the anti-Bax function is also disrupted in GSS-associated PrP mutants and is not associated with the difference between CJD and GSS.