The motivation to perform physical activity is a crucial factor in achieving a persistent active lifestyle. However, motivation decreases with increasing age from childhood to adolescence. The promotion of physical activity in educational settings might be an important tool to increase motivation and, consequently, to decrease sedentary behavior. The aim of this study was to explore the effect of a 4-month school-based walking intervention on motivation to participate in physical activity among Italian middle-school students. This study included 276 students (mean age 13 ± 1 years, 41.3% female). A total of 138 students (intervention group) participated in a brief walking intervention that was added to their routine daily school activity, while a convenience sample of 138 students performed the routine daily school activity. The activity consisted of walking one kilometer outside of school buildings during the morning break. Motivation data were collected before and after the walking breaks using the Participation Motivation Questionnaire (PMQ). Controlling for age, significant interactions between group and time were observed in the "Social Status" [F(1,273) = 4.851; p = 0.028], "Team" [F(1,273) = 6.015; p = 0.015] and "Energy Release" components [F(1,273) = 8.527; p = 0.038]. Specifically, a significant decrease in the "Social Status" components of the PMQ and an increase in the "Team" and "Energy Release" components were observed in the intervention group. On the contrary. control group showed an increase in "Social Status" and a decrease in the "Team" and "Energy Release" components. Within this developmental context, incorporation of the walking activity helped to modify the motivational orientation towards physical activity to include more intrinsic factors, which were related to the possibility of remaining with classmates and peer groups and releasing a surplus of energy.