A new member of the 14-3-3 protein family in Schistosoma mansoni has been identified. Sequence analysis demonstrated that this protein is a member of the epsilon sub-group and is the orthologue of Schistosoma japonicum 14-3-3epsilon. Since we had previously identified a 14-3-3epsilon protein from S. mansoni, we termed the original protein 14-3-3epsilon-1 and this second epsilon protein 14-3-3epsilon-2. Schistosoma mansoni encodes at least four different 14-3-3 isoforms: the two epsilon proteins and 14-3-3 protein 1 and protein 2, which are zeta-like isoforms. Phylogenetic analysis demonstrated the early divergence of the epsilon isoforms, and that schistosome proteins 1 and 2 are among the oldest non-epsilon 14-3-3 proteins yet identified. Schistosoma mansoni 14-3-3epsilon-1, 14-3-3epsilon-2, and protein 1 are stage specifically expressed in a similar manner, being absent in cercariae and schistosomula, and abundant in lung stage and adult male and female worms. Protein 2 transcript was not detected at any of the life cycle stages examined. All three detected 14-3-3 isoforms elicit an immune response during infection, with the greatest response directed against protein 1. Binding studies with S. mansoni receptor kinase-1 (SmRK1) and human Raf kinase revealed that the three 14-3-3epsilon isoforms exhibit a preference for target protein binding. Although all three isoforms do bind to both targets, 14-3-3 protein 1 interacts most strongly with Raf, whereas the 14-3-3-1 isoform binds SmRK1 preferentially. These results suggest that the individual 14-3-3 proteins may have evolved to play isoform-specific roles in the development and survival of S. mansoni within its host.