InMycobacterium tuberculosis, the genes Rv1954A–Rv1957 form an operon that includes Rv1955 and Rv1956 which encode the HigB toxin and the HigA antitoxin respectively. We are interested in the role and regulation of this operon, since toxin–antitoxin systems have been suggested to play a part in the formation of persister cells in mycobacteria. To investigate the function of thehigBAlocus, effects of toxin expression on mycobacterial growth and transcript levels were assessed inM. tuberculosisH37Rv wild type and in an operon deletion background. We show that expression of HigB toxin in the absence of HigA antitoxin arrests growth and causes cell death inM. tuberculosis. We demonstrate HigB expression to reduce the abundance of IdeR and Zur regulated mRNAs and to cleave tmRNA inM. tuberculosis,Escherichia coliandMycobacterium smegmatis. This study provides the first identification of possible target transcripts of HigB inM. tuberculosis.