Background Few studies have investigated associations of acclerometer-based assessments of physical activity (PA) and sedentary behavior (SB) with incidence of cardiovascular disease (CVD) and its components. This prospective cohort study assessed the associations of accelerometer-measured PA and SB with total CVD, myocardial infarction, and ischemic stroke (IS). Methods and Results The authors included 16 031 women aged 62 years and older, free of CVD, with adherent accelerometer wear (≥10 hours/day for ≥4 days) from the Women's Health Study (mean age, 71.4 years [SD, 5.6 years]). Hip-worn ActiGraph GT3X+ accelerometers measured total volume of PA (total average daily vector magnitude), minutes per day of high-light PA and moderate to vigorous PA (MVPA), and SB. Women reported diagnoses of CVD, which were adjudicated using medical records and death certificates. Hazard ratios (HRs) were estimated for each exposure, and 95% CIs using Cox proportional hazards models were adjusted for accelerometer wear time, age, self-reported general health, postmenopausal hormone therapy, smoking status, and alcohol use. The hypothetical effect of replacing 10 minutes/day of SB or high-light PA with MVPA on CVD incidence was assessed using adjusted isotemporal substitution Cox models. Over a mean of 7.1 years (SD, 1.6 years) of follow-up, 482 total CVD cases, 107 myocardial infarction cases, and 181 IS cases were diagnosed. Compared with the lowest quartiles of total average daily vector magnitude and MVPA (≤60 minutes), women who were in the highest quartiles (>120 minutes of MVPA) had a 43% (95% CI, 24%-58%) and 38% (95% CI, 18%-54%) lower hazard of total CVD, respectively. Estimates were similar for total average daily vector magnitude and MVPA with IS, but PA was not associated with myocardial infarction overall. High-light PA was not associated with any CVD outcomes. Women who spent <7.4 hours sedentary per day had a 33% (95% CI, 11%-49%) lower hazard of total CVD compared with those who spent ≥9.5 hours sedentary. Replacing 10 minutes of SB with MVPA was associated with a 4% lower incidence of total CVD (HR, 0.96 [95% CI, 0.93-0.99]). Conclusions Accelerometer-assessed total PA and MVPA were inversely associated with total CVD and IS incidence, and SB was directly associated with total CVD; high-light PA was not related to CVD.