Purpose: The purpose of this study was to determine the 3-year trajectory of students' interest in learning physical activity (PA) knowledge during middle school and the effects of gender and prior knowledge on the trajectory. Methods: Using a repeated-measures design, a cohort of 447 sixth-grade students' interest in learning PA knowledge was measured eight times from the beginning of sixth grade to the end of eighth grade. Students' prior PA knowledge was measured at the beginning of sixth grade. Results: Hierarchical linear modeling analyses showed that students' interest in PA knowledge in general declined over time. Girls' interest declined faster than that of the boys before eighth grade, but their decline slowed during the eighth-grade year. Prior knowledge did not influence the trajectories. Discussion: These findings provide insights regarding students' interest in learning PA knowledge and indicate that prior knowledge may not be a central component that contributes to interest development.