Previous studies indicated that e-books were beneficial for educational settings while animated films also have positive effects on student learning. However, few studies integrated animated films into an e-book. Thus, this study developed an animated e-book, which makes the best use of the advantages of multimedia and online learning to improve student learning. However, recent research suggested that gender differences affected students' attitudes toward multimedia and online learning. Therefore, this study aims to investigate how gender differences influence the use of the animated e-book. The result from this study indicated that gender differences did not exist in the use of the animated e-book. However, correlations between learning perception and learning performance were found for male students while no such significant correlations were shown for females.