Background: In youth, research on the health benefits of breaking up sitting time is inconsistent. Our aim was to explore the association between the number of breaks in sitting time and adiposity in Australian toddlers. Methods: This study comprised 266 toddlers (52% boys), aged 19.6 ± 4.2 months from the GET-UP! Study, Australia. Body mass index (BMI) was calculated and z-scores by age and sex were computed for waist circumference (WC). Participants were classified as overweight according to the WHO criteria for BMI. For WC, participants with a z-score≥1SD were considered overweight. Sitting time was assessed with activPALs during childcare hours and participants were classified by tertiles of the number of breaks/h in sitting time: <26 breaks/h; 26-39 breaks/h, and >39 breaks/h. Logistic regression assessed odds ratios for non-overweight (BMI or waist circumference categories) by number of breaks in sitting time/h, controlling for age, sex, and socioeconomic status. Results: The number of breaks in sitting time significantly predicted a lower weight status (non-overweight) according to WC values (P for trend = 0.032) after adjustments. Conclusions: Breaking up sitting time was positively associated with toddlers’ waist circumference. Future studies are needed to determine whether breaking up sitting time is a protective for cardiometabolic health in toddlers.