Physical education teachers play a significant role in educating students with special education needs, but they often feel frustrated and unprepared to achieve a successful inclusion process. The lack of inadequate training has proven to have an unfavorable effect on educators’ perceived competence, confidence, and attitudes when working in inclusive settings with children with disabilities. This study evaluates the effect of a training program implemented with in-service physical educators from two different regions in Spain, insular and peninsular, in order to improve their self-efficacy in including students with disabilities in physical education. A pre- vs. post-test intervention was conducted with two groups of 142 in-service physical educators with similar age and teaching experience. The intervention improved their perceived self-efficacy in including students with physical, visual, and intellectual disabilities in physical education (p < 0.01, effect sizes = large). The physical educators teaching in the insular region improved their self-efficacy to a higher extent. This study demonstrates the invariant effect of the training program in both regions, but the self-efficacy scores of the physical educators might be constrained by the contents regarding inclusion in their pre-service training, the education policies of the region where the teaching takes place or the geographical characteristics of their workplace.