BACKGROUND: The Eustachian tube is an osteocartilaginous channel connecting the tympanic cavity with the nasopharynx. There is no anatomical and histological research performed on this organ in buffalo. OBJECTIVES: Anatomical and histological study of Eustachian tube in buffalo will be useful for basic knowledge of this organ. METHODS: For this study 8 adult male and female buffalo's head were provided from slaughter house and their Eustachian tube were studied anatomically, then tissue samples were obtained and paraffin sections were prepared for using of staining methods such as H&E (for general study), Verhoff (for elastic fibers), PAS (for carbohydrates) and Masson's Trichrome Stainning Kit (for collagen fibers). RESULTS: Anatomical results showed Eustachian tube was white and funnel- like tube, no curve and structurally supported by cartilage and in both sexes they had the same structure. Histological and Histochemical results showed the epithelium of Eustachian tube in buffalo is pseudostratified ciliated columnar and in some regions of the Eustachian tube epithelium was stratified squamous. In the first portion of Eustachian tube cartilage was elastic and then eustachian tube cartilage was hyaline. The glands of Eustachian tube in buffalo were mucous and non folicular tubal tonsil could be found around the pharyngeal opening with obviously lymphoid tissue. CONCLUSIONS:. The results of this research can be used as the basic anatomical and Histological knowledge in buffalo..