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Introduction Mental health conditions are a major public health challenge. Though typically diagnosed in adolescence, these conditions may arise during early childhood. Considerable evidence shows that engagement in healthy movement behaviours (ie, regular physical activity, reduced sedentary behaviour and sufficient sleep) is associated with a lower risk of mental health conditions in adolescents and adults. However, whether these behaviours have the same protective effect during early childhood is less clear. The MOvement behaviours and irritaBILITY in early childhood (MOBILITY) study was established to investigate the association between 24-hour movement behaviours and trajectories of early childhood mental health.Methods and analysis MOBILITY is a prospective observational cohort study of toddlers’ and preschoolers’ (aged 2–4 years) movement behaviours and mental health, which will be assessed at three data collection points (baseline and 6- and 12-month follow-ups). Mental health indicators (ie, irritability, internalising, externalising and prosocial behaviour) will be assessed via parent-report questionnaires, and movement behaviours will be assessed via ActiGraph accelerometers and sleep diaries. Primary analyses will include using: (1) compositional data analysis to examine prospective relationships between overall movement behaviour compositions and trajectories of mental health indicators, (2) isotemporal substitution modelling to determine whether the prospective association between independent movement behaviours and trajectories of mental health indicators is contingent on other movement behaviours and (3) the ‘Goldilocks’ approach to determine optimal time use compositions to promote favourable trajectories in mental health symptomology.Ethics and dissemination Ethical approval for this study was received from the Health Sciences Research Ethics Board at Western University, London, Ontario, Canada. The findings of this novel research will have implications for informing updates of evidence-based movement behaviour guidelines, as well as the development and implementation of preventive mental health interventions. Study findings will be disseminated via publications and conference presentations for academic audiences, infographics and short videos for community partners, early childhood educators and parents, as well as actionable recommendations for physicians and healthcare providers.