As a result of educational reforms in many countries, including Finland, new or renovated comprehensive schools have increasingly begun to incorporate open and flexible designs and principles. Multipurpose and adaptable open learning spaces can provide children with amplified opportunities to be physically active during general education. Classroom-based physical activity has been associated with better academic-related outcomes and students’ on-task behaviour, while overall physical activity has been associated with better health. In the present study, we investigated the effects of classroom type, gender and grade level on classroom-based physical activity, and the associations between systematically-observed teachers’ instructions about students’ movement and classroom-based physical activity. The participants consisted of 182 3rd and 5th grade students in one school with open learning space and two schools with conventional classrooms. Overall, classroom-based physical activity, assessed with accelerometery, was not higher in open learning space than in conventional classrooms. However, 5th grade students had more sedentary time and less moderate-to-vigorous physical activity in open learning spaces than conventional classrooms, but both 3rd and 5th graders had more breaks from sedentary time in open learning spaces than conventional classrooms. Girls were more sedentary than boys, while 5th graders were less physically active than 3rd graders. Teachers’ instructions regarding 5th graders’ movement in open learning spaces were more restrictive and both 3rd and 5th graders had more instructed transitions in open learning spaces. In conventional classrooms, students had more teacher-organised physical activity. Teachers’ restrictive guidance was associated with less light physical activity, while teachers’ organised physical activity was associated with more moderate-to-vigorous physical activity.