Background: To explore the time trends (2005-2015) of pedometer-determined weekday and weekend physical activity (PA) and obesity prevalence in 4-7-year-old Czech preschool children and changes in proportion of kindergarten vs. leisure-time PA. Methods: The study compared data of two cross-sectional cohorts of preschool children (2005: 92 boys and 84 girls; 2015: 105 boys and 87 girls) in the Czech Republic, using the same measurements and procedures in both cases. PA was monitored by the Yamax Digiwalker SW-200 pedometer for at least eight continuous hours a day over seven consecutive days. Body weight and height were measured using calibrated Tanita scales and anthropometry. The analysis of variance was conducted to examine the gender and cohort effect on step counts. The t-test was used to examine the difference in step counts in kindergarten (or leisure-time) between non-obese and obese children, and the chi-square test compared the prevalence of obesity between 2005 and 2015. Results: The steps/day (mean [plus or minus] standard deviation) of preschoolers was significantly higher (p < 0.05) in 2015 (11,739 [plus or minus] 4,229 steps/day) than in 2005 (10,922 [plus or minus] 3,181 steps/day); and (p < 0.001) in boys (11,939 [plus or minus] 3,855 steps/day) than in girls (10,668 [plus or minus] 3,587 steps/day). In 2015, girls, but not boys, had a significantly (p < 0.01) greater step count on weekdays than in 2005, but not at weekends. A decline of leisure-time step counts on weekdays between 2005 and 2015 in girls (6,865[2005] vs. 6,059[2015], p < 0.01) and boys (7,861[2005] vs. 6,436[2015], p < 0.001) is compensated for by the increase of step counts in kindergarten (girls: 3,058[2005] vs. 5,330[2015], and boys: 4,003[2005] vs. 5,999[2015], p < 0.001). The prevalence of obesity was not significantly different either in 2005 or 2015 among preschool girls (7.14 % [2005] vs. 9.20 % [2015]) or boys (6.52 % [2005] vs. 9.52 % [2015]). Conclusion: The steps/day of preschoolers was higher in 2015 than in 2005; this higher level of PA was the result of increased PA in kindergartens over the last ten years, particularly among girls. Thus, the current PA program in kindergartens effectively compensates for the decline in PA in leisure-time of weekdays of non-obese and obese preschoolers compared to 2005 and 2015. Prevalence of obesity among Czech preschool children remains relatively stable between 2005 and 2015. (author abstract)