Background: This study examined the associations between environmental characteristics of early childhood education and care (ECEC) centers and 1-year change in toddlers’ physical activity and sedentary behavior while at the centers. Methods: Data from 292 toddlers from the GET-UP! study were analyzed. Environmental characteristics of ECEC centers were rated using the Infant/Toddler Environment Rating Scale-revised edition at baseline. Children’s physical activity and sedentary behavior in the centers were assessed using activPAL devices, at baseline and at 1-year follow-up. Linear mixed models were performed to examine the associations between the environmental characteristics and change in the proportion of time spent in physical activity and sedentary behavior. Results: Compared with baseline, children spent a higher proportion of time in sedentary behavior (sitting) but a lower proportion of time in standing and physical activity (stepping) while at ECEC centers, at 1-year follow-up. The environmental characteristics “interaction” (B = −1.39; P = .01) and “program structure” (B = −1.15; P = .04) were negatively associated with change in the proportion of time spent in physical activity. Conclusion: Better “interaction” and “program structure” may preclude children’s physical activity from declining over time and may be considered important features to target in future interventions in ECEC centers aiming at promoting active lifestyles.