Background: The 2018 US Physical Activity Guidelines recommend reducing sedentary behavior (SB) for cardiovascular health. SB’s role in heart failure (HF) is unclear. Methods: We studied 80 982 women in the Women’s Health Initiative Observational Study, aged 50 to 79 years, who were without known HF and reported ability to walk ≥1 block unassisted at baseline. Mean follow-up was 9 years for physician-adjudicated incident HF hospitalization (1402 cases). SB was assessed repeatedly by questionnaire. Time-varying total SB was categorized according to awake time spent sitting or lying down (≤6.5, 6.6–9.5, >9.5 h/d); sitting time (≤4.5, 4.6–8.5, >8.5 h/d) was also evaluated. Hazard ratios and 95% CI were estimated using Cox regression. Results: Controlling for age, race/ethnicity, education, income, smoking, alcohol, menopausal hormone therapy, and hysterectomy status, higher HF risk was observed across incremental tertiles of time-varying total SB (hazard ratios [95% CI], 1.00 [referent], 1.15 [1.01–1.31], 1.42 [1.25–1.61], trend P <0.001) and sitting time (1.00 [referent], 1.14 [1.01–1.28], 1.54 [1.34–1.78], trend P <0.001). The inverse trends remained significant after further controlling for comorbidities including time-varying myocardial infarction and coronary revascularization (hazard ratios: SB, 1.00, 1.11, 1.27; sitting, 1.00, 1.09, 1.37, trend P <0.001 each) and for baseline physical activity (hazard ratios: SB 1.00, 1.10, 1.24; sitting 1.00, 1.08, 1.33, trend P <0.001 each). Associations with SB exposures were not different according to categories of baseline age, race/ethnicity, body mass index, physical activity, physical functioning, diabetes, hypertension, or coronary heart disease. Conclusions: SB was associated with increased risk of incident HF hospitalization in postmenopausal women. Targeted efforts to reduce SB could enhance HF prevention in later life.