Recently, Universal Design for Learning has emerged as an important paradigm for successful inclusive education. In this context, this study aims to investigate and compare the perceptions regarding the significance on Universal Design for Learning between preservice secondary general and special education teachers. Altogether 129 preservice general education teachers and 96 preservice special education teachers in education college training courses in Incheon, Jeollabuk-do, and Gyeongsangbuk-do responded to questionnaires. An independent sample t-test was applied to determine the overall perceptions of Universal Design for Learning and three guidelines. Regarding “providing multiple means of engagement,” preservice secondary special education teachers demonstrated significantly higher perceptions on options for effort, interest, attention, and class engagement than preservice general education teachers. Regarding “providing multiple means of representation,” preservice secondary special education teachers recognized the importance of learning materials, methods, and technology more than preservice general education teachers. Regarding “providing multiple means of action and expression,” preservice secondary special education teachers indicated significantly higher importance on options for providing learning objectives, strategies, motivation, communication expression, and fluency more than preservice general education teachers. Finally, based on the results, implications for the field and suggestions for future research were discussed.