Objective: Physical activity (PA) is critical for healthy development in preschoolers, with long-lasting benefits that can affect later life. The World Health Organization (WHO) recommends that children aged 5–17 years should engage in 60 min of moderate-to-vigorous PA per day. However, physical inactivity in children is on the rise globally, with declines in PA starting at the age of 4 years. Increasing PA during early childhood is important to delay adiposity rebound, promote behavioral changes, improve physical fitness, and facilitate future PA engagement. However, limited evidence has been established on the effects of school-based PA interventions on preschoolers. This study examines the effects and sustainability of a preschool-based PA intervention on increasing PA, improving physical fitness and health in preschoolers, with the exercise dose benchmarked to the WHO PA guidelines. Methods: This assessor-blinded, two-arm cluster randomized controlled trial will include 3300 preschoolers (aged 5–6 years) from 110 kindergartens in Hong Kong, China. Kindergartens will be randomized into intervention and control groups in a 1:1 ratio. The control kindergartens will continue their usual curriculum of ∼2.5 h PA/week, whereas preschoolers in the intervention kindergartens will engage in an additional 75-min game-based PA class twice per week (extra 2.5 h PA/week) over the preschool year. This multi-component intervention will also target parents, teachers, and the kindergarten environment to further encourage PA in preschoolers and their families. Objectively measured PA, cardiorespiratory fitness and other physical fitness components (muscle strength and power, agility, balance, flexibility, body composition), and psychological health will be examined at the start (0 month) and end (10 months) of the preschool year. Maintenance effects will be assessed after preschoolers’ transition into primary school (16 months). Generalized estimating equations or other appropriate