Background Despite growing scientific interest in the benefits of breaking up sedentary time with intermittent standing or walking, few studies have investigated the energy cost of posture transitions. This study aimed to determine whether posture transitions are associated with increased energy expenditure in preschool children. Methods Forty children (mean age 5.3 ± 1.0y) completed a ~150-min room calorimeter protocol involving sedentary, light, and moderate- to vigorous-intensity activities. This study utilised data from ~65-min of the protocol, during which children were undertaking sedentary behaviours (TV viewing, drawing/colouring in, and playing with toys on the floor). Posture was coded as sit/lie, stand, walk, or other using direct observation; posture transitions were classified as sit/lie to stand/walk, sit/lie to other, stand/walk to other, or vice versa. Energy expenditure was calculated using the Weir equation and used to calculate individualised MET and activity energy expenditure (AEE) values. Spearman’s rank correlations were used to compare the number of posture transitions, in the individual activities separately and combined, with corresponding MET and AEE values. Participants were divided into tertiles based on the number of posture transitions; MET and AEE values of children in the lowest and highest tertiles of posture transitions were compared using unpaired t-tests. Effect sizes (Cohen’s d) were calculated. Results There was a positive correlation between the total number of posture transitions and average METs (r s = 0.42, p = 0.02) and AEE (r s = 0.43, p = 0.02). MET differences between the lowest and highest tertiles of posture transitions resulted in a small effect size for playing with toys (d = 0.27), and moderate effect sizes for TV viewing, drawing and all three activities combined (d = 0.61, 0.50 and 0.64 respectively). Similar results were found for AEE. Conclusions Results from this study showed that variation in posture transitions may be associated with variation in energy expenditure in preschool children. The findings suggest that the concept that variation in posture transitions may have meaningful biological or health effects in early childhood is worth investigating further.