Proteasomal stress is believed to contribute to the pathology of ischemic brain injury and several neurodegenerative disorders, but can activate both cytoprotective and cell death-inducing pathways. Here we have utilized the complex environment of organotypic hippocampal slice cultures (OHSCs) to investigate the stress responses activated in different neuronal populations following proteasome inhibition. Incubation of OHSCs with the specific proteasome inhibitors, epoxomicin or bortezomib led to a selective injury of the CA1 pyramidal neurons although similarly increased levels of poly-ubiquitinylated proteins were detected throughout all regions of the hippocampus. Micro-dissection, quantitative PCR and immunohistochemical analyses of epoxomicin-treated OHSCs identified a selective activation of cytoprotective genes in non-vulnerable regions, and a selective activation of p53 target genes within the CA1. Genetic deletion of the pro-apoptotic p53 target gene, p53-upregulated modulator of apoptosis (puma), significantly reduced injury within the CA1 following proteasomal inhibition. Activation of cytoprotective genes by treatment with inducers of heat shock protein 70 inhibited the selective activation of p53 signaling within the CA1 and protected CA1 neurons from epoxomicin-induced cell death. In summary, we demonstrate that the reciprocal activation of p53/p53-upregulated modulator of apoptosis and heat shock protein 70 signalling determines the selective vulnerability of neurons to proteasome inhibition.