Biomphalysins are β-Pore Forming Toxins (β-PFT) identified in the planorbid Biomphalaria glabrata that belong to the aerolysin-like protein family. Despite potentially diverse biochemical activities, very few eukaryotic aerolysin-related proteins have been extensively studied. Most of the data refers to their discovery in genomes or to transcriptional activity. The involvement of biomphalysins in the immune response of Biomphalaria glabrata has been studied previously, especially regarding biomphalysin 1, which can bind and kill Schistosoma mansoni mother sporocysts. However, the repartition of biomphalysin 1 protein in B. glabrata has yet to be defined. The transcriptional behavior of the 22 other biomphalysin genes following immune challenge also remains uncharacterized. Therefore, herein, we investigate for the first time the tissular distribution of biomphalysin 1 (and 2) in B. glabrata by histological and cytological analyses through immunofluorescence approaches, notably unveiling unexpected tissue location that are involved in biomphalysin 1 synthesis. Structural predictions of the 23 members of the family have been updated using predictions based on aminoacyl spatial pair representation (AlphaFold2), highlighting unique features of the small lobe. In addition, mass spectrometry-based proteomic data more precisely predicted the regions of post-translational cleavage of biomphalysin 1. Transcriptional activity of the biomphalysin genes was explored, after which the plasmatic presence of the biomphalysin proteins was investigated in naive and S. mansoni-infected snails. The ability of native biomphalysin 1 (and 2) to bind several cell types was also investigated and correlated with the lytic ability of plasma toward the exposed cells, highlighting the central role occupied by biomphalysin 1 (and 2) in the humoral immunity of B. glabrata.