Bacterial genomes encode a collection of small peptides that are deleterious to their hosts when overexpressed. The physiological relevance of the majority of these peptides is unknown at present, although many of them have been implicated in regulatory processes important for cell survival and adaptability. One peptide that is of particular interest to us is a 19-amino acid proteic toxin, coined IbsC, whose production is repressed by SibC, an RNA antitoxin. Together, IbsC and SibC constitute a type I toxin-antitoxin (TA) pair. To better understand the function of IbsC and to decipher the sequence determinants for its toxic phenotype, we carried out extensive sequence analyses of the peptide. We generated a series of truncation and single amino acid deletion mutants to determine the minimal sequence required for toxicity. We further probed into functionally relevant amino acids with a comprehensive set of IbsC mutants produced using a systematic sequence randomization strategy. We found that IbsC remained toxic in the presence of multiple deletions and single amino acid substitutions, despite being well-conserved in Escherichia coli and across other Gram-negative bacteria. The toxicity of this peptide was determined to be dependent on a stretch of highly hydrophobic residues near its center. Our results defined sequence-function relationship of IbsC and offered additional insights into properties common to membrane-targeting type I toxins in E. coli and related species.