Microsporidia are a group of obligate intracellular parasites causing significant disease in human beings and economically important animals. Though a few spore wall proteins (SWPs) have now been identified in these intriguing species, the information on SWPs remains too little to elucidate the spore wall formation mechanisms of microsporidia. It has been well described that numerous proteins with tandem repeats tend to be localized on the cell wall of fungi and parasites. Previously, by scanning the proteins with tandem repeats in microsporidian Nosema bombycis, we obtained 83 candidate SWPs based on whether those proteins possess a signal peptide and/or transmembrane domain. Here, we further characterized a candidate protein (EOB13250) with three tandem repeats in the N-terminal region and a transmembrane domain in C-terminus of N. bombycis. Sequence analysis showed that the tandem repeat domain of EOB13250 was species-specific for this parasite. RT-PCR indicated that the expression of the gene encoding this protein started on the fourth day postinfection. After cloned and expressed in Escherichia coli, a polyclone antibody against the recombinant EOB13250 protein was prepared. Western blotting demonstrated this protein exist in N. bombycis. Immunofluorescence analysis (IFA) and immunoelectron microscopy analysis (IEM) further provided evidence that EOB13250 was an endospore wall protein. These results together suggested that EOB13250 was a novel spore wall protein of N. bombycis. This study provides a further enrichment of the number of identified spore wall proteins in microsporidia and advances our understanding of the spore wall formation mechanism in these obligate unicellular parasites.