Background: Prolonged sitting is considered detrimental to health, but evidence regarding the independent relationship of total sitting time with all-cause mortality is limited. This study aimed to determine the independent relationship of sitting time with all-cause mortality. Methods:Welinked prospective questionnaire data from 222 497 individuals 45 years or older from the 45 and Up Study to mortality data from the New South Wales Registry of Births, Deaths, and Marriages (Australia) from February 1, 2006, through December 31, 2010. Cox proportional hazards models examined all-cause mortality in relation to sitting time, adjusting for potential confounders that included sex, age, education, urban/rural residence, physical activity, body mass index, smoking status, self-rated health, and disability. Results: During 621 695 person-years of follow-up (mean follow-up, 2.8 years), 5405 deaths were registered. Allcause mortality hazard ratios were 1.02 (95% CI, 0.95- 1.09), 1.15 (1.06-1.25), and 1.40 (1.27-1.55) for 4 to less than 8, 8 to less than 11, and 11 or more h/d of sitting, respectively, compared with less than 4 h/d, adjusting for physical activity and other confounders. The population- attributable fraction for sitting was 6.9%. The association between sitting and all-cause mortality appeared consistent across the sexes, age groups, body mass index categories, and physical activity levels and across healthy participants compared with participants with preexisting cardiovascular disease or diabetes mellitus. Conclusions: Prolonged sitting is a risk factor for allcause mortality, independent of physical activity. Public health programs should focus on reducing sitting time in addition to increasing physical activity levels.