Background: In this study, we examined moderate-to-vigorous physical activity (MVPA) of children in a school district serving children from low-income and minority households. Methods: This observational study was conducted in 8 rural elementary schools in South Carolina. Children (N = 719, age = 7.7 years, 48.0% girls, 88.0% African American) wore accelerometers during school hours. Physical activity was distilled into time engaged in MVPA during 4 distinct opportunities. These 4 opportunities were non-activity time (eg, class-time), extended lunch (lunch recess after eating), physical education (PE), and recess. Mixed effects linear regressions estimated MVPA on days that had no activity opportunities, extended lunch, recess, PE, and multiple activity opportunities (eg, PE and recess). Results: On days with multiple activity opportunities, girls and boys accumulated 8.0 (95% CI = 4.9, 11.1) and 7.1 (95% CI = 3.6, 10.7) additional minutes of MVPA compared to a no activity day. On PE days boys accumulated 5.2 (95% CI = 0.3, 10.2) additional minutes of MVPA, whereas recess days provided girls with 3.0 (95% CI = 0.1, 6.0) additional MVPA minutes. No other activity opportunities provided statistically significant increases in school-day MVPA. Conclusions: In low-income schools it may be necessary to provide multiple physical activity opportunities during the school day to increase boys' and girls' MVPA.