The transition from kindergarten to school is associated with a variety of negative changes. After entry to elementary school physical activity level decreases. Moreover, physical fitness level of children over the past decades have rapidly declined. Children are spending an increasing amount of time in the environments that require constant sitting. We evaluated the differences between boys and girls in physical fitness, frequency of undertaking of different forms of physical activity, prevalence of underweight and overweight, and time spent on sedentary behavior. A total of 212 first grade pupils (mean age 6.95 ± 0.43) from two standard urban schools in Poznań participated in the study. Compared to girls, boys obtained better results in 20-meter run (4.9 s and 5.0 s, p < 0.01), sit-ups (16.8 and 15.3, p < 0.05), six-minute run (829.7 m and 766.4 m, p < 0.001), and standing broad jump (106.8 cm and 99.7 cm, p < 0.01). In the sit-and-reach test girls achieved higher results than boys (17.0 cm and 14.4 cm, p < 0.001). There were no gender differences in prevalence of underweight and overweight. In conclusions, difference between genders should be taken into consideration during designing physical activity programs in the aspects of intensity and forms of physical activities.Author Contributions: Conceptualization, P.L., M.B. and A.K.; formal analysis, P.L., M.B. and A.K.; funding, M.B.; investigation, P.L., M.B. and A.K.; methodology, P.L., M.B. and A.K.; validation, P.L., M.B. and A.K.; writing-original draft preparation, P.L., M.B. and A.K.; writing-review and editing, P.L., M.B. and A.K. All authors contributed to the development of the research and the writing of the article. All authors have read and agreed to the published version of the manuscript.