Preservice teachers should learn about motivation theory during their university studies in order to be able to motivate their students in their future job. Thus, this study had two objectives. First, it explored whether experience in education sciences and a motivation theory seminar were related to preservice teachers' perceived experience with motivation theory, their perceived and actual knowledge about it, and the relevance they assigned to it. Second, it investigated whether there were differences to practicing teachers. We administered a questionnaire on three motivational theories to N = 322 to preservice and practicing teachers. We asked for the participants' experience with the topics, the relevance they assigned to them in the school context, and their perceived knowledge about them. Overall, preservice teachers rated motivation theory as relevant but displayed low experience with and knowledge about it. Visiting a seminar on motivation theory was connected to the highest values for all variables. Practicing teachers appeared more critical of motivation theory than preservice teachers. They assigned less relevance to it, although they displayed higher levels in perceived knowledge than preservice teachers without a motivation theory seminar background. Results suggest that there is a difference between the preservice and the practicing phase of teaching regarding motivation theory. Preservice teachers viewed the topic differently than practicing teachers. However, preservice teachers should know and be able to use motivation theory once they finish their university studies. Therefore, integrating motivation theory more into both preservice and practicing teacher training appears to be important.