By adopting Sport Education into the university context, this study examined how lesson content and teacher interaction contribute to university students’ physical activity levels during physical education lessons. Following a continuing professional development workshop, a 10-week Sport Education season was designed collaboratively and implemented at a university, taking into account the university’s facilities. This study used the momentary time sampling tool, System for Observing Fitness Instruction Time, in which eight teachers and 202 students (72% male) were videotaped in 156 lessons. The data on physical activity levels, lesson content, and teacher behavior were collected and analyzed. The student-participants engaged in an average of 38.77 (±18.78) moderate-to-vigorous physical activity (MVPA) minutes which did not meet the recommendation of 50% lesson time. Findings of hierarchical linear regression indicated that knowledge (β = 0.29) and general content (β = 0.29) contributed to the sitting physical activity while skill practices (β = 0.25) and gameplay (β = 0.38) predicted the MVPA. Practical implications of utilizing the features of Sport Education in boosting the intensity of activities are discussed, especially team affiliation, formal competition, and record keeping. Further investigations are proposed on the interaction of gameplay content and teacher behavior with larger sample size.