This study investigates the association between participation in structured multisport activities at different weekly frequencies and gross motor development in preschool children, aiming to provide practical evidence for promoting gross motor skills in early childhood. A total of 63 preschool children were classified into four groups according to their weekly participation frequency in structured multisport activities: the low-frequency group (L; up to 1 session/week, with intermittent participation; n = 16), the medium-frequency group (M; fixed participation in 1 session/week, with occasional participation in a second session; n = 15), the medium-high-frequency group (MH; fixed participation in 2 sessions/week, with occasional participation in a third session; n = 18), and the high-frequency group (H; fixed participation in ≥ 3 sessions/week; n = 14). All groups participated in a 12-week structured multisport activity program (60 minutes per session). Gross motor development was assessed before and after the intervention using the Test of Gross Motor Development-3 (TGMD-3). A 2 (Time: pre- vs. post-intervention) × 4 (Group: L/M/MH/H) mixed-design ANOVA was applied to evaluate changes in each gross motor development outcome within and between groups. After the 12-week intervention, (1) the total score of gross motor development showed a significant Time × Group interaction (p < 0.01, η²p = 0.280). All groups improved (p < 0.01), and H, MH, and M showed greater improvements than L (p < 0.05). (2) The total score of locomotor movements showed a significant Time × Group interaction (p < 0.01, η2p = 0.205). All groups improved (p < 0.01), and H and MH showed greater improvements than L (p < 0.05). For individual locomotor skills, horizontal jump showed a significant interaction (p < 0.05, η²p = 0.143); all groups improved (p < 0.01), and H and MH showed greater improvements than L (p < 0.05). (3) The total score of object control movements showed a significant Time × Group interaction (p < 0.01, η2p = 0.183). All groups improved (p < 0.01), and H and MH showed greater improvements than L (p < 0.05). For individual object control skills, two-hand strike of a stationary ball, one hand stationary dribble and two hand catch showed significant Time × Group interactions (p < 0.05, η²p = 0.124, 0.128, and 0.163, respectively). For two-hand strike of a stationary ball and one hand stationary dribble, H and MH showed greater improvements than L (p < 0.05), whereas the significant interaction for two hand catch did not translate into significant Bonferroni-adjusted between-group differences at either pre-test or post-test. (4) The L group did not show significant improvements in gallop, skip, and one hand stationary dribble (p > 0.05), whereas the other groups improved in a wider range of movements (p < 0.05). After three months of intervention, preschool children participating in structured multisport activities showed improvements in overall gross motor development. Regular participation (once a week or more) was associated with improvement across all assessed gross motor skills. More frequent participation (at least twice per week) was associated with greater improvements in specific skills, including horizontal jump, two-hand strike of a stationary ball, and one hand stationary dribble. Although gross motor development generally tended to improve as participation frequency increased, no statistically significant additional advantage of the high-frequency group over the medium-high-frequency group was detected. These findings should be interpreted cautiously, given the absence of a non-intervention control group and the relatively limited sample size.

• Regular participation in structured multisport activities (once a week or more) was associated with improvements in all assessed gross motor skills.

• Participation in at least two sessions per week was associated with greater improvements in specific skills, including horizontal jump, two-hand strike of a stationary ball, and one hand stationary dribble.

• No statistically significant additional advantage of the high-frequency group over the medium-high-frequency group was detected.