School-based physical activity benefits the health and well-being of children and youth, but students’ activity levels are different. The aim of this study is to evaluate the intervention effect of a school-based physical activity intervention on Health-related Quality of Life for groups with different physical activity profiles at baseline. The intervention, Move for Well-being in School, targeted PE lessons, in-class activities, and physical activities during recess. It was evaluated in a cluster-randomized design and included students from 4 to 6th grade at 24 Danish public schools. Survey data on Health-related Quality of Life (KIDSCREEN-27), physical activity variables and socio-demographics were collected prior to intervention and after 9 months. The students were grouped by a latent class analysis using responses on physical activity behavior and physical self-perception: most active and most confident, moderately active and moderately confident, and least active and least confident. Students in the least active group were more likely to be member of a family from lower social class and have an overweight body image. The multilevel analyses for the (combined) associations of time, intervention and student group showed that the effect of the intervention was different between groups. For the KIDSCREEN dimensions physical well-being, psychological well-being, autonomy and parent relation showed significant interactions (p < 0.1) in favor of the lesser active and confident students in the intervention group. Trial registration: Date of registration: 24th of April 2015 retrospectively registered at Current Controlled Trials with Study ID ISRCTN12496336.