BackgroundThe ShK toxin fromStichodactyla helianthushas established the therapeutic potential of sea anemone venom peptides, but many lineage-specific toxin families in Actiniarians remain uncharacterised. One such peptide family, sea anemone 8 (SA8), is present in all five sea anemone superfamilies. We explored the genomic arrangement and evolution of the SA8 gene family inActinia tenebrosaandTelmatactis stephensoni, characterised the expression patterns of SA8 sequences, and examined the structure and function of SA8 from the venom ofT.stephensoni.ResultsWe identified ten SA8-family genes in two clusters and six SA8-family genes in five clusters forT. stephensoniandA. tenebrosa, respectively. Nine SA8T. stephensonigenes were found in a single cluster, and an SA8 peptide encoded by an inverted SA8 gene from this cluster was recruited to venom. We show that SA8 genes in both species are expressed in a tissue-specific manner and the inverted SA8 gene has a unique tissue distribution. While the functional activity of the SA8 putative toxin encoded by the inverted gene was inconclusive, its tissue localisation is similar to toxins used for predator deterrence. We demonstrate that, although mature SA8 putative toxins have similar cysteine spacing to ShK, SA8 peptides are distinct from ShK peptides based on structure and disulfide connectivity.ConclusionsOur results provide the first demonstration that SA8 is a unique gene family in Actiniarians, evolving through a variety of structural changes including tandem and proximal gene duplication and an inversion event that together allowed SA8 to be recruited into the venom ofT.stephensoni.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12915-023-01617-y.