Spermatogenesis is a complex process of terminal differentiation by which mature sperms are generated,and it can be divided into three phases:mitosis,meiosis and spermiogenesis.In a previous study,we established a series of proteomic profiles for spermatogenesis to understand the regulation of male fertility and infertility.Here,we further investigated the localization and the role of flotillin-2 in spermiogenesis.Flotillin-2 expression was investigated in the testis of male CD1 mice at various developmental stages of spermatogenesis by using Western blotting,immunohistochemistry and immunofluorescence.Flotillin-2 was knocked down in vivo in three-week-old male mice using intratesticular injection of small inhibitory RNA(siRNA),and sperm abnormalities were assessed three weeks later.Flotillin-2 was expressed at high levels in male germ cells during spermatogenesis.Flotillin-2 immunoreactivity was observed in pachytene spermatocytes as a strong dot-shaped signal and in round spermatids as a sickle-shaped distribution ahead of the acrosome.Immunofluorescence confirmed flotillin-2 was localized in front of the acrosome in round spermatids,indicating that flotillin-2 was localized to the Golgi apparatus.Knockdown of flotillin-2 in vivo led to a significant increase in head sperm abnormalities isolated from the cauda epididymis,compared with control siRNA-injected testes.This study indicates that flotillin-2 is a novel Golgi-related protein involved in sperm acrosome biogenesis. Spermatogenesis is a complex process of terminal differentiation by which mature sperms are generated, and it can be divided into three phases: mitosis, meiosis and spermiogenesis. In a previous study, we established a series of proteomic profiles for spermatogenesis to understand the regulation of male fertility and infertility. Here, we further investigated the localization and the role of flotillin-2 in spermiogenesis. Flotillin-2 expression was investigated in the testis of male CD1 mice at various developmental stages of spermatogenesis by using Western blotting, immunohistochemistry and immunofluorescence. Flotillin -2 was knocked down in vivo in three-week-old male mice using intratesticular injection of small inhibitory RNA (siRNA), and sperm abnormalities were assessed three weeks later. Flotillin-2 was expressed at high levels in male germ cells during spermatogenesis. Flotillin-2 immunoreactivity was observed in pachytene spermatocytes as a strong dot-shaped signal and in round spermatids as a sickle-shaped distribution ahead of the acrosome. Immunofluorescence confirmed that flotillin-2 was localized in front of the acrosome in round spermatids, indicating that flotillin-2 was localized to the Golgi apparatus. Knockdown of flotillin-2 in vivo led to a significant increase in head sperm abnormalities isolated from the cauda epididymis, compared with control siRNA-injected testes. This study indicates that flotillin-2 is a novel Golgi-related protein involved in sperm acrosome biogenesis.