Vibrio cholerae codes for 13 toxin–antitoxin (TA) loci all located within the superintegron on chromosome II. We show here that the two higBA TA loci of V. cholerae encode functional toxins, HigB-1 and HigB-2, whose ectopic expression inhibits cell growth of Escherichia coli, and functional antitoxins, HigA-1 and HigA-2, which counteract the toxicity of the cognate toxins. Three hours of ectopic expression of the HigB toxins resulted in bacteriostasis without any detectable loss of cell viability. The HigB toxins inhibited translation by cleavage of mRNA. Efficient mRNA cleavage occurred preferentially within the translated part of a model mRNA and only when the mRNA was translatable. Promoter analysis in V. cholerae and E. coli showed that the two higBA loci are both transcribed into bi-cistronic mRNAs and that the higBA-2 mRNA is leaderless. Transcription of the two higBA loci was strongly induced by amino acid (aa) starvation in V. cholerae and E. coli, indicating that the regulatory mechanisms of transcriptional induction are conserved across the two species. Both higBA loci stabilized a test-plasmid very efficiently in E. coli, raising the possibility that the loci contribute to maintain genetic stability of the V. cholerae superintegron. Based on these results we discuss the possible biological functions of the TA loci of V. cholerae.