Adequate energy intake is essential for the healthy development of children, and the estimated energy requirement of children is determined by total daily energy expenditure (TDEE) and energy deposition for growth. A previous study in Japanese tweens indicated that TDEE could be estimated by fat-free mass (FFM) and step count. The aims of this study were to measure TDEE in Japanese preschool children and to confirm whether TDEE can be estimated by FFM and step count in preschool children. Twenty-one children aged 4-6 years old (11 girls and 10 boys; age, 5.1 (0.9) years; height, 107.2 (6.6) cm; weight, 17.5 (1.7) kg; BMI, 15.3 (1.3); mean (SD)) participated in this study. FFM and 7-day TDEE were obtained by doubly labeled water (DLW). Participants wore accelerometers during the DLW measurement period. No significant differences were observed in age-adjusted height, weight, BMI, FFM (13.0 (1.5) kg), or TDEE (1300 (174) kcal/day) between girls and boys. Girls had significantly higher percent fat and a lower daily step count than boys. Stepwise regression analysis revealed that FFM and step count were significant predictors of TDEE; TDEE (kcal/day) = 85.0 × FFM (kg) + 0.0135 × step count (steps/day). This accounted for 74% of TDEE variance. The current study confirmed that FFM and step count are major determinants of TDEE in Japanese preschool children as well as adolescents, although further research is needed to obtain precise equations.