Background: Rett syndrome (RTT) is a rare neurodevelopmental disorder leading to multiple disabilities and high dependency on caregivers. This study aimed to: (1) describe the patterns of sedentary time and daily steps and (2) identify the association of individual and environmental characteristics with sedentary time. Methods: All Danish females with RTT older than 5 years of age and with a MECP2 mutation were invited to participate. The activPAL and StepWatch Activity Monitor (SAM) were worn by participants for at least four days. Sedentary time and step counts were plotted by time to examine daily activity patterns. Associations between sedentary time and individual and environmental covariates were assessed with linear regression models. Results: The median (interquartile range) age of participants was 22.0 (14.3–36.5) years. On average 83.3% (standard deviation 13.9%) of waking hours were spent in sedentary behaviours (n = 48) and the median (interquartile range) daily step count was 5128 (2829–7704) (n = 28). Females older than 33.5 years, and those unable to walk independently were more sedentary. Conclusions: This study demonstrated high levels of sedentary time and low daily step counts in a Danish population of females with RTT. Advancing age and lower walking skills were associated with higher levels of sedentary time.Implications for RehabilitationSedentary lifestyles in individuals with disabilities have a negative impact on health and quality of life.High levels of sedentary time and low daily step counts were demonstrated in a Danish population of females with Rett syndrome.Advancing age and inability to walk independently were strongly associated with higher levels of sedentary time in females with Rett syndrome.Understanding patterns of sedentary behaviour and physical activity can aid health care professionals in developing health-promoting physical activity interventions.