Afterschool programs are an important setting in which to promote children's physical activity. This study examines the association of environmental and policy characteristics on the moderate-to-vigorous physical activity and sedentary behavior of children attending afterschool programs. Methods: A total of 1302 children attending 20 afterschool programs across South Carolina wore accelerometers (ActiGraph GT3X+) for up to 4 non-consecutive days. Policy-level characteristics were evaluated using the Healthy Afterschool Program Index-Physical Activity scale. Physical activity space was measured using a measuring wheel (indoor, ft[squared]) and Geographical Information Systems software (outdoor, acres). The structure (free-play or organized) of activity opportunities was evaluated via direct observation. Time spent in moderate-to-vigorous physical activity and sedentary, both indoors and outdoors, was estimated using accelerometry. Results: For every 5000 ft[squared] of utilized indoor activity space an additional 2.4 and 3.3 min/day of sedentary behavior was observed among boys and girls, respectively. A higher ratio of free-play to organized play was associated with higher indoor sedentary behavior among boys and girls (3.9 min/day and 10.0 min/day, respectively). For every 1 acre of outdoor activity space used, an additional 2.7 min/day of moderate-to-vigorous physical activity was observed for boys. A higher free-play to organized play ratio was associated with higher outdoor moderate-to-vigorous physical activity for boys and girls (4.4 and 3.4 min/day increase, respectively). Policy characteristics were unrelated to moderate-to-vigorous physical activity levels and time spent sedentary. Conclusion: Findings indicate that policies and size of activity space had limited influence on moderate-to-vigorous physical activity and sedentary behavior, suggesting that a programmatic structure may be a more effective option to improve moderate-to-vigorous physical activity levels of children attending afterschool programs. (author abstract)