The purpose of this study was to determine the effect of stand-biased desks on the physical activity and sedentary behavior of third, fourth and sixth grade students across the school year. Methods: This within classroom crossover design study used teacher-determined allocation for seating within each classroom. Half of the students used a stand-biased desk and half used a sitting desk. Five-day hip-worn accelerometer assessments were completed at baseline and at the end of each nine-week intervention period. A mixed effects model was used to determine the differences in the percentage of time spent active and sedentary. Results: A total of 22, 36 and 41 students in 3rd, 4th and 6th grades, respectively, completed this study (57.1% male, 79.3% White). Regardless of the desk type, students became more sedentary (p < 0.001) and less active (p < 0.001) in the classroom as the school year progressed. After controlling for baseline activity, there was a significant interaction between the type of desk and time (p = 0.029). Students who spent a higher percentage of their classroom time sedentary engaged in less sedentary behavior when using a stand-biased desk compared to the traditional desk. Conclusion: The standing desk intervention was effective in mitigating the increase in sedentary behavior for those who started the school year more sedentary.