Abstract: In order to explore the morphology and ultrastructure of Gentoo penguin（Pygoscelis papua） spermatozoa, the fresh semen were collected from five male Gentoo penguins in breeding season by non-invasive and consistent back massage, and the spermatozoa were analyzed by scanning electron microscopy（SEM） and transmission electron microscopy（TEM）,respectively. The results showed that the morphology of the Gentoo penguin spermatozoa by SEM were typically flagellated, with an ellipsoid head, 11.40～11.60 μm long, a relatively shorter midpiece, 1.83～2.69 μm long, and a slender and long tail, 47.50～59.74 μm long. The ultrastructure of Gentoo penguin spermatozoa by TEM showed that the head of Gentoo penguin spermatozoa was long, narrow and ellipsoid, the tail was elongated and flagellated.9+2 microtubule structure, 0.30～0.42 μm long, was found parallel to the central axis of the tail. The autophagosome, 0.61～0.81 μm long, was found in the midpiece. This study putatively elucidated the morphology and ultrastructure of sperm in Gentoo penguins, intended to provide a foundation for studying the fertilization mechanism and the construction of reproduction, and germplasm resource bank for Gentoo penguins, and a technical support for preservation of genetic diversity, ex situ conservation of Gentoo penguins.