A wide range of biomolecules, including proteins, are excreted/secreted from helminths and contribute to the parasite-s successful establishment, survival and reproduction in an adverse habitat. Excretory/secretory proteins (ESP) are active at the interface between parasite and host and comprise potential targets for intervention. The intestinal nematode Strongyloides spp. exhibits an exceptional developmental plasticity in its life cycle characterized by parasitic and free-living generations. We investigated ESP from infective larvae (iL3), parasitic females (pF) and free-living stages (flS) of the rat parasite Strongyloides ratti,which is genetically very similar to the human pathogen, Strongyloides stercoralis. Proteomic analysis of ESP revealed 586 proteins, with largest number of stage-specific ESP found in iL3 (196), followed by pF (79) and flS (35). One hundred and forty proteins were identified in all studied stages, including anti-oxidative enzymes, heat shock proteins, and carbohydrate-binding proteins. The stage-selective ESP of (i) iL3 included an astacin metalloproteinase, the L3 Nie antigen and a fatty acid retinoid-binding protein; (ii) pF included a prolyl oligopeptidase (prolyl serine carboxypeptidase), small heat shock proteins, and a secreted acidic protein; (iii) flS included a lysozyme family member, a carbohydrate-hydrolysing enzyme, and saponin-like protein. We verified the differential expression of selected genes encoding ESP by qRT-PCR. ELISA analysis revealed the recognition of ESP by antibodies of S. ratti-infected rats. A prolyl oligopeptidase was identified as abundant pF-specific ESP, and the effect of pyrrolidine-based prolyl oligopeptidase inhibitors showed concentration- and time-dependent inhibitory effects on female motility. The characterization of stage-related ESP from Strongyloides will help to further understand the interaction of this unique intestinal nematode with its host.