PURPOSE: To examine the associations between participation frequency in Physical Education (PE) classes and objective measures of physical activity (PA) and sedentary behavior (SB) in children from 12 countries at different levels of development. METHODS: This multinational, cross-sectional study included 5,874 children aged 9-11 years from sites in Australia, Brazil, Canada, China, Colombia, Finland, India, Kenya, Portugal, South Africa, the United Kingdom and the United States. PA and SB were monitored over 7 consecutive days using a waist-worn accelerometer. PA and SB data were presented for weekdays (times in- and out-of-school) and weekend days. Participation frequency in PE classes was determined by questionnaire. Multilevel modeling analyses stratified by sex were used. RESULTS: Overall, 24.8% of children self-reported participation in PE classes ≥ 3 times/week (25.3% in high-income countries [HIC], and 24.3% in low- and middle-income countries [LMIC]). After adjusting for age, sex, parental education and body mass index z-score, results showed that children from LMIC who took PE classes 1-2 times/week were more likely to present better indicators of PA and shorter time in SB in- and out-of-school. In HIC, boys that participated in PE classes were more likely to meet the moderate-to-vigorous PA (MVPA) recommendations and to present better indicators of PA (in school) and shorter time in SB in- and out-of-school. For girls in HIC, attending PE classes increased the likelihood of spending more time in MVPA, especially if they attended ≥ 3 times/week. CONCLUSION: Attending PE classes is associated with a higher level of PA and lower level of SB in- and out-of-school during weekdays in children from countries at various levels of development.