Background: High levels of sedentary behavior are often measured in preschoolers, but only a few interventions have been developed to counteract this. Furthermore, detailed descriptions of interventions in preschoolers targeting different forms of sedentary behavior could not be located in the literature. The aim of the present paper was to describe the different steps of the Intervention Mapping Protocol used towards the development of an intervention component of the ToyBox-study focusing on decreasing preschoolers' sedentary behavior. The ToyBox-study focuses on the prevention of overweight in 4- to 6-year-old children by implementing a multi-component kindergarten-based intervention with family involvement in six different European countries. Methods: Applying the Intervention Mapping Protocol, six different steps were systematically completed for the structured planning and development of the intervention. A literature search and results from focus groups with parents/caregivers and kindergarten teachers were used as a guide during the development of the intervention and the intervention materials. Results: The application of the different steps in the Intervention Mapping Protocol resulted in the creation of matrices of change objectives, followed by the selection of practical applications for five different intervention tools that could be used at the individual level of the preschool child, at the interpersonal level (i.e., parents/caregivers) and at the organizational level (i.e., kindergarten teachers). No cultural differences regarding preschoolers' sedentary behavior were identified between the participating countries during the focus groups, so cultural and local adaptations of the intervention materials were not necessary to improve the adoption and implementation of the intervention. Conclusions: A systematic and evidence-based approach was used for the development of this kindergarten-based family-involved intervention targeting preschoolers, with the inclusion of parental involvement. The application of the Intervention Mapping Protocol may lead to the development of more effective interventions. The detailed intervention matrices that were developed as part of the ToyBox-study can be used by other researchers as an aid in order to avoid repetitive work for the design of similar interventions.