Flipped learning is a pedagogical approach that directs instruction from a group to an individual learning space. This approach can stimulate students' motivation, promote adequate physical activity levels, and reduce sedentary (SED) behaviour. Addressing the literature gap regarding the correlations among these factors in school physical education (PE), this study aimed to examine the association of students' motivation, physical activity levels, and SED behaviour with flipped learning in a four-lesson learning unit. Framed by self-determination theory, a quasi-experimental design was adopted to measure and compare the moderate-to-vigorous physical activity (MVPA), SED, and motivation levels of 111 primary school students aged between 10 and 11 years (mean = 10.07, standard deviation = 0.26) in Hong Kong. Fifty-two of the participants were female, and 59 were male. They were randomly assigned to experimental (n = 57) and control (n = 54) groups, in which a flipped learning approach was adopted in the experimental group for two weeks. A questionnaire survey and accelerometers were used to measure participants' motivation (autonomous motivation, controlled motivation, and amotivation) and their physical activity levels. The findings revealed significantly higher MVPA levels and lower SED levels in the experimental group. Autonomous motivation was found to have a positive correlation with MVPA levels and a negative correlation with SED levels, indicating a positive predictor of the two. The findings of this study reveal the potential of flipped learning to enhance students' MVPA and reduce their SED behaviour during primary school PE lessons.