Male obesity is associated with reduced sperm function and increased incidence of sperm DNA damage; however, the underlying molecular mechanisms have not yet been identified. Mammalian SIRT6 protein is involved in caloric-dependant DNA damage repair in other tissue types, yet a possible role for SIRT6 in male obesity and subfertility has not been investigated previously. To assess SIRT6 levels and activity in the testes, male mice (n=12 per diet) were fed either a control diet (CD; 6% fat) or a high-fat diet (HFD; 21% fat) for 16 weeks before the collection of testes and spermatozoa. SIRT6 protein was localised to the nucleus of transitional spermatids and the acrosome of mature spermatozoa, with levels significantly decreased in HFD-fed male mice (P<0.05). This decrease in SIRT6 protein was associated with transitional spermatids having increased levels of acetylated H3K9 in the nucleus (P<0.01) and increased DNA damage (P<0.001). We propose a role for SIRT6 in spermiogenesis and potentially protamination processes, which are known to be compromised by male obesity.