Little is known concerning the type of activity that should be substituted for sedentary time and its potentially most hazardous form (prolonged sedentary bouts) to impart health benefit. We used isotemporal substitution techniques to examine whether 1) replacing total sedentary time with light-intensity or moderate to vigorous physical activity (LIPA or MVPA) or 2) replacing prolonged sedentary bouts with shorter sedentary bouts is associated with reductions in all-cause mortality risk. Participants (n = 7,999) from the Reasons for Geographic and Racial Differences in Stroke (REGARDS) study, a national cohort of US adults aged ≥45 years, were studied. Sedentary time was measured by accelerometry between 2009 and 2013. There was a beneficial association with mortality risk for replacing total sedentary time with both LIPA (per 30 minutes, hazard ratio (HR) = 0.83; 95% confidence interval (CI): 0.80, 0.87) and MVPA (per 30 minutes, HR = 0.65; 95% CI: 0.50, 0.85). Similarly, there was a beneficial association for replacing prolonged sedentary-bout time with LIPA and MVPA but not for replacement with shorter sedentary bouts (per 30 minutes, HR = 1.00; 95% CI: 0.96, 1.03). These findings suggest short sedentary bouts still carry mortality risk and are not a healthful alternative to prolonged sedentary bouts. Instead, physical activity of any intensity is needed to mitigate the mortality risks incurred by sedentary time.