Objective: To determine the association between standing time and all-cause mortality. Methods: Prospective questionnaire data from 221,240 individuals from the 45 and Up Study were linked to mortality data from the New South Wales Registry of Deaths (Australia) from February 1, 2006 to June 17, 2012. Hazard ratios for all-cause mortality according to standing time at baseline were estimated in 2013 using Cox regression modelling, adjusted for sex, age, education, urban/rural residence, physical activity, sitting time, body mass index, smoking status, self-rated health and disability. Results: During 937,411 person years (mean follow-up = 4.2. yr) 8009 deaths occurred. All-cause mortality hazard ratios were 0.90 (95% CI 0.85-0.95), 0.85 (95% CI 0.80-0.95), and 0.76 (95% CI 0.69-0.95) for standing 2-≤ 5 h/d, 5 - ≤ 8. h/d, or > 8. h/d respectively, compared to standing two or less hours per day. Further analyses revealed no significant interactions between standing and sex (p = 0.93), the presence/absence of cardiovascular disease or diabetes (p = 0.22), BMI (p = 0.78), physical activity (p = 0.16) and sitting time (p = 0.22). Conclusion: This study showed a dose-response association between standing time and all-cause mortality in Australian adults aged 45. years and older. Increasing standing may hold promise for alleviating the health risks of prolonged sitting.