The purpose of this study was to evaluate the efficacy of need-supportive teaching in physical education on girls' daily moderate-to-vigorous physical activity using a mixed method evaluation. 507 sixth-grade girls aged 9–14 years of 33 single-sex physical education classes participated in the cluster randomized control trial. During the 16-week intervention period, trained teachers conducted enhanced physical education lessons which were designed based on self-determination theory. In a randomized process, independent researchers using a computer-based algorithm allocated classes to the trial groups (IG n = 19 classes, CG n = 14). These lessons were subject to repeated systematic observations. The students' perceptions of basic psychological need support and satisfaction in physical education were measured using repeated self-report questionnaires. Students' daily moderate-to-vigorous physical activity (MVPA) was assessed by accelerometry. Semi-structured interviews provided a deeper understanding of how purposively sampled focus groups perceived teacher behavior in physical education. After a separate analysis of qualitative and quantitative data, results were merged to investigate the intervention's efficacy and treatment fidelity. Throughout the school year, the girls' MVPA levels decreased in both groups. Girls who reported their complete physical activity data had a lower body mass index than girls who reported no, or only one or two sets of physical activity data. Results of mixed measures converge on the finding that the teachers in the intervention group provided slightly stronger need support than the control teachers, however, intervention components were not delivered consistently. Therefore, a significant intervention effect on daily MVPA could not be quantified. Autonomy satisfaction significantly predicted MVPA. Qualitative insights of teaching behavior in PE underlined the importance of need support and revealed structural barriers, which compromised the implementation quality. Ethics Committee of the Technical University of Munich 155/16S; Bavarian Ministry of Education IV.8-BO6106/52/12. German Research Foundation grant DE2680/3-1. •Sixth-grade girls perceive and appreciate a need-supportive teaching behavior in physical education.•PE teachers' influence on girls' daily moderate-to-vigorous physical activity is limited.•Accelerometer-measured physical activity levels of girls decrease within one school year significantly.