Importance: Sedentary behavior is associated with several health outcomes, including diabetes, cardiovascular disease, and all-cause mortality. Less is known about the association between objectively measured sedentary behavior and cancer mortality, as well as the association with physical activity. Objective: To examine the association between accelerometer-measured sedentary behavior (total volume and accrual in prolonged, uninterrupted bouts) and cancer mortality. Design, Setting, and Participants: A prospective cohort study conducted in the contiguous US included 8002 black and white adults aged 45 years or older enrolled in the Reasons for Geographic and Racial Differences in Stroke (REGARDS) study. The present analysis was performed from April 18, 2019, to April 21, 2020. Exposures: Sedentary time, light-intensity physical activity (LIPA), and moderate- to vigorous-intensity physical activity (MVPA) were measured using a hip-mounted accelerometer worn for 7 consecutive days. Main Outcomes and Measures: Cancer mortality. Results: Of the 8002 study participants, 3668 were men (45.8%); mean (SD) age was 69.8 (8.5) years. Over a mean (SD) follow-up of 5.3 (1.5) years, 268 participants (3.3%) died of cancer. In multivariable-adjusted models, including MVPA, greater total sedentary time was associated with a greater risk of cancer mortality (tertile 2 vs tertile 1: hazard ratio [HR], 1.45; 95% CI, 1.00-2.11; tertile 3 vs tertile 1: HR, 1.52; 95% CI, 1.01-2.27). Longer sedentary bout duration was not significantly associated with greater cancer mortality risk: after adjustment for MVPA (tertile 2 vs tertile 1: HR, 1.26; 95% CI, 0.90-1.78; tertile 3 vs tertile 1: HR, 1.36; 95% CI, 0.96-1.93). Replacing 30 minutes of sedentary time with LIPA was significantly associated with an 8% (per 30 minutes: HR, 0.92; 95% CI, 0.86-0.97) lower risk of cancer mortality; MVPA was significantly associated with a 31% (per 30 minutes: HR, 0.69; 95% CI, 0.48-0.97) lower risk of cancer mortality. Conclusions and Relevance: In this cohort study, greater sedentary time, as measured with accelerometry, appeared to be independently associated with cancer mortality risk. Replacing sedentary time with either LIPA or MVPA may be associated with a lower risk of cancer mortality. These findings suggest that the total volume of sedentary behavior is a potential cancer mortality risk factor and support the public health message that adults should sit less and move more to promote longevity.