OBJECTIVES: The purpose of this observational study was to examine differences in preschoolers' sedentary time and physical activity (PA) participation between preschool-attending weekdays with and without a teacher-led structured PA session. DESIGN: A sample of 200 preschoolers (5.3±0.4y; 113 boys) from 26 preschools in Flanders, Belgium were included in data analysis. METHODS: Participants wore a GT1M ActiGraph accelerometer on one preschool-attending weekday with and on one preschool-attending weekday without the provision of a teacher-led structured PA session. Preschoolers' sedentary time, light PA, and moderate-to-vigorous physical activity (MVPA) during the time in preschool (08:00-16:00h) and after preschool (16:00-20:00h) were estimated. To assess differences in the outcome measures between both days, multi-level linear regression models were conducted. RESULTS: During the time at preschool, lower sedentary levels (β=13.0min; SE=1.6; p<0.001) and higher light PA (β=2.9min; SE=0.7 p<0.001) and MVPA levels (β=10.1min; SE=1.1; p<0.001) were prevalent on days with a structured PA session compared to days without a structured PA session in both boys and girls. After preschool, no differences were found between both days in sedentary time (β=0.7; SE=1.4; p>0.05), light PA (β=0.3; SE=0.5; p>0.05), and MVPA (β=0.3; SE=0.9; p>0.05). CONCLUSIONS: The results demonstrate that no compensatory changes were found after preschool for the structured PA session during the preschool hours. Therefore, a teacher-led structured PA session integrated in the preschool curriculum is a promising mean to decrease sedentary time and to increase PA in preschool-aged boys and girls.