Being active offers many physical and emotional benefits contributing to a higher health-related quality of life (HRQoL); however, this relationship remains unexplored among preschoolers (aged 2.5–5 years). This study examined the impact of the Supporting Physical Activity in the Childcare Environment (SPACE), which was an intervention implemented using a cluster randomized controlled trial on preschoolers’ HRQoL. Childcare centres were randomly allocated to the experimental (n = 11) or control (n = 11) conditions, and preschoolers’ HRQoL was measured using the parent-report Pediatric Quality of Life Inventory 4.0 (3 subscales: physical, psychosocial, and total HRQoL) at baseline, post-intervention (i.e., week 8), and 6- and 12-month follow-up. A linear mixed-effects model was used to determine if preschoolers in the experimental condition displayed an increased HRQoL post-intervention and at follow-up compared with preschoolers in the control condition. Preschoolers (n = 234) with HRQoL data at baseline and one additional time-point were retained for analyses. Body mass index was not found to impact significantly on the intervention, and no statistically significant interaction effects were found for any of the 3 HRQoL variables. In conclusion, the SPACE intervention had no impact on preschoolers’ HRQoL. Given the scarcity of research in this population, additional exploration is necessary to better understand the potential impact of physical activity participation on preschoolers’ HRQoL.