Abstract: The enteric microsporidiosis of hatchery-bred juvenile grouper, Epinephelus spp., is the most important in the mariculture area off coast of South China Sea in recent years, however, the taxonomy of the causative agent remains unknown. In this study, histopathological, ultrastructural and molecular evidences were provided to identify the aetiological agent, designated herein as Enterospora epinepheli sp. n.. The intranuclear development of the present species was consistent with Enterospora canceri, the type species of Enterospora genus. The early stages of uninucleate meronts were observed within the infected nuclei, separating from the host nucleus by a simple electron dense membrane. Later, the uninucleate meronts transformed into multinucleate plasmodia (merogonial plasmodia). At this stage, the infected nuclei were hypertrophic, or even ruptured by the multinucleate plasmodia. Sporogonial plasmodia were characterized by the appearance of multiple, small, spherical, membrane-bound vesciles. Then, multiple copies of these membrane-bound vesciles developed into the precursors of the polar filament and anchoring disk of mature spore which surrounded the diplokaryotic nuclei. With the development, sporoblasts separated from the plasmodia by successive division and direct development to mature spores. Mature spores were oval, in direct contact with host nucleus. Spores measured 1.56±0.31 (1.07—1.96) μm in length and 1.08±0.98 (0.93—1.28) μm in width. The spore walls were trilaminar, including an electron dense exospore coat 15.51±0.95 nm (9.87—26.18 nm) thick, surrounding a thick electron lucent endospore (81.13±2.71) nm (57.16—110.81 nm) thick and the plasma membrane. The polar filaments were isofilar, coiled with 5—6 turns in two rows. Histopathological analysis clearly revealed that the spores located in the nucleus of goblet cell of the intestinal epithelial and a large amount of spores appeared in the intestinal contents with the shedding necrotic infected enterocytes. Molecular analysis indicated that genetic distances ofEnterospora epinepheli sp. n. form the species of Enterocytozoondiae ranged from 0.162 to 0.225 which was generally out of intraspecies variation. Phylogenetic analysis revealed that the species of Enterocytozoondiae could separate into Clade Ⅰand Clade Ⅱ, and Enterospora epinepheli sp. n. was an independent lineage, clustering with Enterospora hepatopenaei, E. nucleophilia, E. canceri and Enterocyozoon bieneusi within the Clade Ⅱ.