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This study aimed to determine the causative agent of black spots which observed in the mesentery, adipose tissue and peritoneum of orange-spotted grouper, Epinephelus coioides. The parasitological examination using light and electron microscope confirmed infection with trichosomoidid parasite, genus Huffmanela, which identified as Huffmanela japonica (H. japonica). The examined eggs were present in two stages (stage 1, mature light brown eggs with larva; stage 2, advanced dark brown eggs with grown larva). The histopathological alteration of the infected tissues together with determination of immunological gene expression in different organs demonstrated prominent response of the host tissues against the studied parasite. Pathological examination of the infected fish revealed that the lesions were mainly in the mesenteric and diffuse pancreatic tissues which present in the abdominal fat. Granulomas containing eggs and mononuclear and eosinophilic granular cell infiltrations, together with fat necrosis of the pancreatic tissue, were the most common pathological findings. Migrating larvae and peritoneal adhesions with organised fibrosis were also observed. In the infected tissues as well as in the spleen, liver and kidney, interleukin 1β (IL-1β) and cyclooxygenase (COX)-2 were upregulated than in control non-infected fish. Our results confirmed that H. japonica is infecting orange-spotted grouper caught from the Red Sea in Egypt with prominent tissue specificity in the mesentery, adipose tissue surrounding the intestine, and in the peritoneum. [ABSTRACT FROM AUTHOR]