Significance The stability of the proteome is essential to cellular and organismic health and lifespan. To maintain proteostasis, cells are equipped with a network of chaperones that support folding of nascent proteins, as well as refolding of unfolded or misfolded proteins. Aging and age-associated diseases progressively increase the accumulation of misfolded, damaged, and aggregated proteins, thus taxing the chaperoning machinery to its limits. Here, we describe how AMPylation of cytosolic heat shock proteins (HSP) leads to a collapse of proteostasis, the induction of a strong heat shock response, inhibition of translation, as well as the formation of protein aggregates. AMPylation-mediated inhibition of HSP70 may represent a strategy for targeted ablation of this chaperone.