<div><p>Following the finding that ammodytoxin (Atx), a neurotoxic secreted phospholipase A<sub>2</sub> (sPLA<sub>2</sub>) in snake venom, binds specifically to protein disulfide isomerase (PDI) <i>in vitro</i> we show that these proteins also interact in living rat PC12 cells that are able to internalize this group IIA (GIIA) sPLA<sub>2</sub>. Atx and PDI co-localize in both differentiated and non-differentiated PC12 cells, as shown by fluorescence microscopy. Based on a model of the complex between Atx and yeast PDI (yPDI), a three-dimensional model of the complex between Atx and human PDI (hPDI) was constructed. The Atx binding site on hPDI is situated between domains b and b’. Atx interacts hPDI with an extensive area on its interfacial binding surface. The mammalian GIB, GIIA, GV and GX sPLA<sub>2</sub>s have the same fold as Atx. The first three sPLA<sub>2</sub>s have been detected intracellularly but not the last one. The models of their complexes with hPDI were constructed by replacement of Atx with the respective mammalian sPLA<sub>2</sub> in the Atx—hPDI complex and molecular docking of the structures. According to the generated models, mammalian GIB, GIIA and GV sPLA<sub>2</sub>s form complexes with hPDI very similar to that with Atx. The contact area between GX sPLA<sub>2</sub> and hPDI is however different from that of the other sPLA<sub>2</sub>s. Heterologous competition of Atx binding to hPDI with GV and GX sPLA<sub>2</sub>s confirmed the model-based expectation that GV sPLA<sub>2</sub> was a more effective inhibitor than GX sPLA<sub>2</sub>, thus validating our model. The results suggest a role of hPDI in the (patho)physiology of some snake venom and mammalian sPLA<sub>2</sub>s by assisting the retrograde transport of these molecules from the cell surface. The sPLA<sub>2</sub>–hPDI model constitutes a valuable tool to facilitate further insights into this process and into the (patho)physiology of sPLA<sub>2</sub>s in relation to their action intracellularly.</p></div
