RelE of Escherichia coli is a global inhibitor of translation that is activated by nutritional stress. Activation of RelE depends on Lon-mediated degradation of RelB, the antagonist that neutralizes RelE. In vitro, RelE cleaves synthetic mRNAs positioned at the ribosomal A-site. We show here that in vivo overexpression of RelE confers cleavage of mRNA and tmRNA in their coding regions. RelE-mediated cleavage depended on translation of the RNAs and occurred at both sense and stop codons. RelE cleavage of mRNA and tmRNA was also induced by amino acid starvation. An ssrA deletion strain was hypersensitive to RelE, whereas overproduction of tmRNA counteracted RelE toxicity. After neutralization of RelE by RelB, rapid recovery of translation required tmRNA, indicating that tmRNA alleviated RelE toxicity by rescuing ribosomes stalled on damaged mRNAs. RelE proteins from Gram-positive Bacteria and Archaea cleaved tmRNA with a pattern similar to that of E. coli RelE, suggesting that the function and target of RelE may be conserved across the prokaryotic domains.