This study examined the initial effects of a participatory-based, competency-/skill-building professional development workshop for physical education (PE) teachers on the use of physical activity (PA) promotion practices (e.g., eliminating lines, small-sided games) and students' moderate-to-vigorous physical activity (MVPA). A total of 823 students (52.8% boys) wore accelerometers at baseline (fall 2015) and outcome (spring 2016) on PE and non-PE days. The System for Observing Fitness Instruction Time+ measured changes in PA promotion practices. Teachers (n = 9) attended a 90-minute workshop prior to outcome data collection. Mixed-model linear regressions estimated changes in teacher practices and students' MVPA. Three of the nine targeted PA promotion practices changed in the desired direction (i.e., p < 0.05; increased motor content and lessons taught outdoors, reduced activities with lines), with three more teacher practices trending in the desired direction (i.e., reduced management time and activities with elimination, increased small-sided games). During PE, boys and girls increased MVPA by 2.0 (95% confidence interval [1.1, 3.0]), and 1.3 (95% confidence interval [0.5-2.0]) minutes, respectively. However, there were no statistically significant changes in boys' or girls' MVPA during the school day. Greater implementation of promotion practices by the PE teachers was associated with boys', but not girls', MVPA during PE. Girls in high- and low-implementing teachers' lessons experienced increases in MVPA, suggesting that even small changes in PA promotion practices can increase girls' MVPA during PE. Overall, the workshops were effective at increasing teachers' PA promotion and students' MVPA in PE. Other school-based strategies that complement and extend efforts targeting PE are recommended to increase children's total daily PA.