This is the first report of a functional toxin–antitoxin (TA) locus in Piscirickettsia salmonis. The P. salmonis TA operon (ps-Tox-Antox) is an autonomous genetic unit containing two genes, a regulatory promoter site and an overlapping putative operator region. The ORFs consist of a toxic ps-Tox gene (P. salmonis toxin) and its upstream partner ps-Antox (P. salmonis antitoxin). The regulatory promoter site contains two inverted repeat motifs between the −10 and −35 regions, which may represent an overlapping operator site, known to mediate transcriptional auto-repression in most TA complexes. The Ps-Tox protein contains a PIN domain, normally found in prokaryote TA operons, especially those of the VapBC and ChpK families. The expression in Escherichia coli of the ps-Tox gene results in growth inhibition of the bacterial host confirming its toxicity, which is neutralized by coexpression of the ps-Antox gene. Additionally, ps-Tox is an endoribonuclease whose activity is inhibited by the antitoxin. The bioinformatic modelling of the two putative novel proteins from P. salmonis matches with their predicted functional activity and confirms that the active site of the Ps-Tox PIN domain is conserved.