Background <p>Physical fitness in preschoolers, encompassing muscular strength, speed-agility, balance, and cardiorespiratory fitness, serves as a key health indicator. While preschools are ideal settings for promoting physical fitness, the association between preschool-based movement behaviors and physical fitness remains unclear.</p> Methods <p>This cross-sectional study included 1144 Chinese preschoolers aged 3–6 years. Preschool-based movement behaviors including moderate-to-vigorous physical activity (MVPA), light physical activity (LPA), and sedentary behavior (SB), were measured using ActiGraph GT9X accelerometers. Physical fitness was assessed via the PREFIT battery, which includes handgrip strength, standing long jump, 4 × 10 m shuttle run, one-leg stance, and 20 m shuttle run. Compositional linear regression and isotemporal substitution modeling were employed to examine associations and time-reallocation effects, respectively.</p> Results <p>Greater amounts of MVPA during preschool hours were positively associated with better performance in muscular strength, speed-agility, and cardiorespiratory fitness. Reallocating time from SB or LPA to MVPA enhanced physical fitness, whereas substituting MVPA with SB or LPA reduced fitness levels, demonstrating an asymmetric effect.</p> Conclusions <p>Moderate-to-vigorous physical activity during preschool hours significantly enhances physical fitness. Prioritizing the implementation of physical activity programs to increase MVPA in preschool settings is crucial for improving physical fitness and addressing insufficient MVPA in this age group.</p> Impact <p><UnorderedList Mark="Bullet"> <ItemContent> <p>First large-scale study (<i>N</i> = 1144) demonstrating preschool-based moderate-to-vigorous physical activity (MVPA) is essential for developing preschoolers’ muscular strength, speed-agility, and cardiorespiratory fitness, complementing existing 24-h movement behavior research.</p> </ItemContent> <ItemContent> <p>Reveals critical asymmetry: Reducing MVPA time significantly harms fitness, with losses exceeding the benefits from equivalent MVPA increases.</p> </ItemContent> <ItemContent> <p>Provides objective evidence to guide policymakers in optimizing preschool schedules to prioritize MVPA for enhancing children’s physical fitness.</p> </ItemContent> </UnorderedList></p>