Physical activity may be influenced by one's physical environment, including day length and weather. Studies of physical activity, day length, and weather have primarily used self-reported activity, broad meteorological categorization, and limited geographic regions. We aim to examine the association of day length and physical activity in a large cohort of older women, covering a wide geographic range. Participants (N = 16,741; mean (SD) age = 72.0 (SD = 5.7) years) were drawn from the Women's Health Study and lived throughout the United States. Physical activity was assessed by accelerometer (ActiGraph GT3X+) between 2011 and 2015. Day length and weather information were obtained by matching weather stations to the participants' location using National Oceanic and Atmospheric Administration databases. Women who experienced day lengths greater than 14 hours had 5.5% more steps, 9.4% more moderate-to-vigorous physical activity, and 1.6% less sedentary behavior, compared to women who experienced day lengths less than 10 hours, after adjusting for age, accelerometer wear, temperature, and precipitation. Day length is associated with physical activity and sedentary behavior in older women, and needs to be considered in programs promoting physical activity as well as in the analyses of accelerometer data covering wide geographic regions.