There is a lack of research on the relationship between defecation habits (DF) and physical activity (PA) in preschoolers. This study aimed to clarify the relationship between sedentary behaviour (SB), PA time, and DF in preschoolers and to estimate the effect of DF replacement in an isotemporal substitution (IS) model. The participants included 166 children (aged 4–6 years) attending childcare facilities. PA was measured using an accelerometer to calculate the daily activity and wearing time for SB, light-intensity physical activity (LPA), and moderate-to-vigorous-intensity physical activity (MVPA). DF were classified based on defecation frequency and timing. A multinomial logistic regression analysis was used for the IS model. One-way analysis of variance detected significant differences in MVPA between the DF groups (F(2) = 3.12,p< 0.05). According to the analysis results of the IS model, replacing 5 min of SB with MVPA resulted in improved DF (odds ratio [OR], 0.89; 95% confidence interval [CI], 0.81–0.97). Conversely, replacing 5 min of MVPA with SB worsened DF (OR, 1.13; 95% CI, 1.03–1.23). The findings suggest that PA is associated with DF among preschoolers. It also indicates that replacing SB with MVPA could help improve DF in children.