Although physical education (PE) provides a school-based platform for the enhancement of learners’ physical health, implementation challenges can have a detrimental effect on learners’ motivation to participate in physical activities and their fitness levels. Within the framework of the Self-determination Theory (SDT), meeting learners’ basic psychological needs of autonomy, competence and relatedness in the PE class, can promote their fitness levels by enhancing their intrinsic motivation to be physically active. The purpose with this study was to investigate the effect of an in-service PE teacher training programme including needs-support teaching strategies on the physical and motor fitness levels of the learners of the participating teachers. Using a pre- and post-test experimental design, the fitness of 1 control and 4 experimental groups were assessed using standardised tests before and after the intervention programme. The intervention included implementing the needs-support teaching strategies acquired by the teachers during the once-off, 5-day teacher training programme, for 4 months while receiving continued support from the instructors of the course during those 4 months. The results show that the programme had a positive effect on the fitness levels of the learners in most of the tested fitness components, warranting the recommendation of SDT-based in-service training of PE teachers to support learners’ motivation towards physical activity and fitness.