Objectives. SOFIT+ is an observation tool to measure teacher practices related to moderate-to-vigorous physical activity (MVPA) promotion during physical education (PE). The objective of the study was to examine the validity of SOFIT+ during high school PE lessons. Design. This cross-sectional, observational study tested the construct validity of SOFIT+ in boys' and girls' high school PE lessons. Methods. Twenty-one PE lessons were video-recorded and retrospectively coded using SOFIT+. Students wore hip-mounted accelerometers during lessons as an objective measure of MVPA. Multinomial logistic regression was used to estimate the likelihood of students engaging in MVPA during different teacher practices represented by observed individual codes and a combined SOFIT+ index-score. Results. Fourteen individual SOFIT+ variables demonstrated a statistically significant relationship with girls' and boys' MVPA. Observed lesson segments identified as high MVPA-promoting were related to an increased likelihood of girls engaging in 5-10 (OR = 2.86 [95%CI 2.41-3.40]), 15-25 (OR = 7.41 [95%CI 6.05-9.06]), and 30-40 (OR = 22.70 [95%CI 16.97-30.37]) seconds of MVPA. For boys, observed high-MVPA promoting segments were related to an increased likelihood of engaging in 5-10 (OR = 1.71 [95%CI 1.45-2.01]), 15-25 (OR = 2.69 [95%CI 2.31-3.13]) and 30-40 (OR = 4.26 [95%CI 3.44-5.29]) seconds of MVPA. Conclusions. Teacher practices during high school PE lessons are significantly related to students' participation in MVPA. SOFIT+ is a valid and reliable tool to examine relationships between PE teacher practices and student MVPA during PE.