Prion protein (PrP) prevents Bax-mediated cell death by inhibiting the initial Bax conformational change that converts cytosolic Bax into a pro-apoptotic protein. PrP is mostly a glycophosphatidylinositol-anchored cell surface protein but it is also retrotranslocated into cytosolic PrP (CyPrP) or can become a type 1 or type 2 transmembrane protein. To determine the form and subcellular location of the PrP that has anti-Bax function, we co-expressed various Syrian hamster PrP (SHaPrP) mutants that favour specific PrP topologies and subcellular localization with N-terminally green fluorescent protein tagged pro-apoptotic Bax (EGFP-Bax) in MCF-7 cells and primary human neurons. Mutants that generate both CyPrP and secreted PrP ((Sec)PrP) or only CyPrP have anti-Bax activity. Mutants that produce (Ctm)PrP or (Ntm)PrP lose the anti-Bax activity, despite their ability to also make (Sec)PrP. Transmembrane-generating mutants do not produce CyPrP and both normal and cognate mutant forms of CyPrP rescue against the loss of anti-Bax activity. (Sec)PrP-generating constructs also produce non-membrane attached (Sec)PrP. However, this form of PrP has minimal anti-Bax activity. We conclude that CyPrP is the predominant form of PrP with anti-Bax function. These results imply that the retrotranslocation of PrP encompasses a survival function and is not merely a pathway for the proteasomal degradation of misfolded protein.