Total body water (TBW) measured by isotope dilution techniques can be used to assess body composition safely and accurately in children. Unfortunately, this method is not readily available for most research projects, particularly when working with large groups of people, because the equipment is complicated and highly specialized. Bioelectrical impedance (BI) method is a simple, quick, and inexpensive method for the assessment of total body water (TBW). In Japanese child population, however, a lack of prediction equations is a problem to determine TBW. The purpose of this study was to determine the prediction equation for TBW determination in Japanese children using the isotope dilution technique as the reference method. Seventy Japanese children (39 boys, 31 girls) with ages ranging between 3 and 6 years participated in this study. They were randomly divided into the validation group (26 boys, 20 girls) and cross-validation group (13 boys, 11 girls). In a forward stepwise regression analysis, 96% of the variability in TBW measured by deuterium oxide (D₂O) dilution could be predicted by the following equation: TBW(kg)=0.149×Resistance Index (Stature²/resistance, cm²/Ω)+0.244×Weight(kg)+0.460×Age(y)+0.501×Sex (boy=1, girl=0)+1.628, with a root mean square error (RMSE) of 0.440 kg in the validation group. This equation predicted TBW in the cross-validation group with R²=0.946 and a pure error (PE)=0.400 kg TBW. Hence, this equation should be applicable for predicting TBW in Japanese children aged 3–6 y.