<jats:p>The lantibiotic lacticin 481 is a bacteriocin produced by <jats:italic>Lactococcus lactis</jats:italic> ssp. <jats:italic>lactis</jats:italic>. This polypeptide contains 27 amino acids, including the unusual residues dehydrobutyrine and the thioether‐bridging lanthionine and 3‐methyllanthionine. Lacticin 481 belongs to a structurally distinct group of lantibiotics, which also include streptococcin A‐FF22, salivaricin A and variacin. Here we report the first complete structure of this type of lantibiotic. The exact location of the thioether bridges in lacticin 481 was determined by a combination of peptide chemistry, mass spectrometry and NMR spectroscopy, showing connections between residues 9 and 14, 11 and 25, and 18 and 26.