Pebrine is a serious disease of the silkworm, Bombyx mori, caused by the first identified microsporidium Nosema bombycis, which is an obligate parasitic single-celled eukaryote. The pathogen can spread both horizontally and vertically, severely affecting sericulture. SNARE proteins mainly mediate the transport of vesicles and membrane fusion, playing a key role in the biological processes. The microsporidium is known to have a well-developed membrane system, especially the polaroplast which occupies most of the volume of mature spores. In order to explore the function of microsporidian SNARE protein, the transcription and subcellular localization characteristics of a novel Syntaxin-like protein (NbSTX-like) from N. bombycis that had a conserved t-SNARE motif were analyzed. In the different development stages of N. bombycis, the NbSTX-like expressed in the nucleus of meronts, then transited to the cytoplasm in the sporonts, gradually gathered at the two ends of the sporoblasts, and finally concentrated at the polaroplast, posterior vacuole and plasma membrane region of mature spores. Interestingly, the rNbSTX-like protein could fuse liposomes to form large vesicular and tubular structures. The formation of sporoplasms was inhibited by the anti-NbSTX-like serum, implying that NbSTX-like protein participated in sporoplasm maturation. These findings laid a foundation for studying the function of SNARE proteins in microsporidia and provided new insights for the prevention and control of sericulture pathogens.