Eukaryotic PIN (PilT N-terminal) domain proteins are ribonucleases involved in quality control, metabolism and maturation of mRNA and rRNA. The majority of prokaryotic PIN-domain proteins are encoded by the abundant vapBC toxin--antitoxin loci and inhibit translation by an unknown mechanism. Here we show that enteric VapCs are site-specific endonucleases that cleave tRNA(fMet) in the anticodon stem-loop between nucleotides +38 and +39 in vivo and in vitro. Consistently, VapC inhibited translation in vivo and in vitro. Translation-reactions could be reactivated by the addition of VapB and extra charged tRNA(fMet). Similarly, ectopic production of tRNA(fMet) counteracted VapC in vivo. Thus, tRNA(fMet) is the only cellular target of VapC. Depletion of tRNA(fMet) by vapC induction was bacteriostatic and stimulated ectopic translation initiation at elongator codons. Moreover, addition of chloramphenicol to cells carrying vapBC induced VapC activity. Thus, by cleavage of tRNA(fMet), VapC simultaneously may regulate global cellular translation and reprogram translation initiation.