The objective of the study was to examine long summer vacation (LSV) effect on physical activity (PA) in 68 children from three Slovenian cities. PA was assessed over five consecutive days using accelerometer in 6- to 8-year-old children (N = 68; 35 girls and 33 boys, mean age 7.0 ± 0.8 years). PA was assessed on two occasions, before and after LSV and showed clear Gaussian distribution model. PA dropped after LSV for boys (9.6%, P = 0.004) and girls (9%, P = 0.014). Furthermore, PA intensity changed significantly after LSV. In boys inactivity increased after LSV from 69% to 72.2% (P = 0.009) and in girls from 71.7% to 74.2% (P = 0.031), while light PA decreased from 26.9% to 24% (P = 0.005) in boys and from 24.6% to 22.5% (P = 0.035) in girls. In 27% of boys and in just 13% of girls the amount of physical activity in June also determined the amount of physical activity in September. Furthermore, we found a significant drop in physical activity only in boys (P = 0.002) and girls (P < 0.001) with higher initial (June 2007) values. In conclusion we could state that there is a clear negative effect of long summer vacation on physical activity habits and needs further investigations for clear explanation.