OBJECTIVE: To examine the association among 24-hour movement guidelines and fundamental motor skills (FMS) in preschoolers. DESIGN: Prospective cohort study with one-year follow-up. METHODS: Parents of 3–4-year-old children reported child age, sex, race, and time spent viewing screens (hours/day). Accelerometers measured preschooler physical activity (PA) and sleep. The 24-hour movement guidelines were examined (≥3 hours/day total PA including ≥1 hour/day of moderate-to-vigorous [MVPA], 10–13 hours/day of sleep, and ≤1 hour/day of screen-time). Trained researchers administered the Test of Gross Motor Development-Third Edition (TGMD-3) to assess FMS. Linear regression was used to assess guidelines met and raw locomotor, ball skills, and total TGMD-3 scores with adjustment for covariates. RESULTS: One hundred and seven preschoolers provided complete cross-sectional data, and 53 preschoolers provided complete longitudinal data. Of the 107 preschoolers, they were 3.4±0.6 years of age, 44% were male, 50% were White, and the mean age-and-sex adjusted TGMD-3 total score percentile was below average (41±22). Many preschoolers met the PA guideline (91%) and sleep guideline (83%), but few preschoolers met the screen-time guideline (8%) or all three guidelines (6%). MVPA was positively associated with locomotor and total TGMD-3 scores in cross-sectional analysis (p