Microsporidia are obligate intracellular parasites related to fungi with resistant spores against various environmental stresses. The rigid spore walls of these organisms are composed of two major layers, which are the exospore and the endospore. Two spore wall proteins (the endosporal protein-SWP30 and the exosporal protein-SWP32) have been previously identified in Nosema bombycis. In this study, using the MALDI-TOF-MS technique, we have characterised a new 25.7-kDa spore wall protein (SWP26) recognised by monoclonal antibody 2G10. SWP26 is predicted to have a signal peptide, four potential N-glycosylation sites, and a C-terminal heparin-binding motif (HBM) which is known to interact with extracellular glycosaminoglycans. By using a host cell binding assay, recombinant SWP26 protein (rSWP26) can inhibit spore adherence by 10%, resulting in decreased host cell infection. In contrast, the mutant rSWP26 (rDeltaSWP26, without HBM) was not effective in inhibiting spore adherence. Immuno-electron microscopy revealed that this protein was expressed largely in endospore and plasma membrane during endospore development, but sparsely distributed in the exospore of mature spores. The present results suggest that SWP26 is a microsporidia cell wall protein that is involved in endospore formation, host cell adherence and infection in vitro. Moreover, SWP26 could be used as a good prospective target for diagnostic research and drug design in controlling the silkworm, Bombyx mori, pebrine disease in sericulture.