This study aimed to describe participation and health-related quality of life (HRQoL) in adolescents and young adults with myelomeningocele and to explore their relationships with lifestyle-related factors. Fifty-one individuals with a mean age of 21 years 1 month (SD 4y 6mo) years participated (26 males, 25 females; 82% hydrocephalus, 55% wheelchair-dependent). Participation was assessed using the Life Habits Questionnaire, and HRQoL was assessed using the Medical Outcomes Study 36-item Short-form Health Survey. Physical activity was measured using an accelerometry-based activity monitor, fitness (peak oxygen uptake) was measured during a maximal exercise test, and the sum of four skin-folds was assessed to indicate body fat. Relationships were studied using logistic regression analyses. Of the participants, 63% had difficulties in daily activities and 59% in social roles. Participants perceived lower physical HRQoL than a Dutch reference population. Participants with higher levels of physical activity and fitness had fewer difficulties in participating in daily activities (odds ratio [OR]=8.8, p=0.02 and OR=29.7, p=0.02 respectively) and a higher physical HRQoL (OR=4.8, p=0.02 and OR=30.2, p=0.006 respectively), but not mental HRQoL. Body fat was not related to participation or HRQoL. In conclusion, a large proportion of individuals with myelomeningocele had difficulties in participation and perceived low physical HRQoL. Higher levels of physical activity and fitness were related to fewer difficulties in participation and higher physical HRQoL.