SIRT6, a nuclear protein, has been implicated in a number of essential cellular processes, such as the DNA damage response, metabolic homeostasis, inflammation, tumorigenesis and aging. However, the role of Sirt6 in the regulation of spermatogenesis is yet unknown. In the present study, we successfully generated Sirt6-/- mice on a C57BL6/ICR mixed background and found that some Sirt6-/- mice survived beyond eight weeks. We further revealed that spermatogenesis in Sirt6-/- mice was arrested at the elongated spermatid stage. Sirt6-/- male mice were completely infertile and had an increased number of apoptotic spermatids. To our surprise, deacetylation activities of SIRT6 on H3K9ac, H3K18ac and H3K56c were not required for spermatogenesis. Therefore, our findings establish a novel link between Sirt6 and male fertility, suggesting an essential role of Sirt6 in spermatogenesis.